人SLPI cDNA的克隆及在毕赤酵母中的表达
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摘要
SLPI能抑制多种蛋白水解酶对组织的损伤,同时可抑制多种微生物的生长繁殖,甚至病毒的复制。它对维护局部黏膜组织、防止炎性损伤起到天然平衡作用,是人体自然免疫系统重要的防御分子。
目前已可由重组DNA技术得到SLPI,但因成本高尚未广泛用于临床。从人粘膜分泌液中提取SLPI,存在原料来源有限、病毒不易灭活和纯度不高等问题。市场上重组人SLPI是以大肠杆菌为表达载体的,不但具有原核表达系统永远无法超越真核表达系统的缺陷,而且SLPI蛋白的阳离子特性能会对大肠杆菌产生毒害作用并影响其生存繁殖,所以无法实现高表达,难以满足医学研究的需要。目前尚未见有用毕赤酵母表达SLPI蛋白的研究报道。鉴于此,本研究选用毕赤酵母作为SLPI蛋白生产的生物反应器,开展以下工作:
1.本研究构建克隆载体pBS-SLPI,经测序证明序列正确,含有完整的人SLPI基因。以此为基础,可以通过重组改造实现病毒载体基因治疗,也可以进行下一步重组蛋白的研究。
2.本研究构建表达载体pPICZα-SLPI,经测序证实含有SLPI正确读框结构。用毕赤酵母表达SLPI蛋白经15% SDS-PAGE电泳分析,在蛋白质分子量12kDa附近可见一条考马斯亮蓝染色蛋白区带,与文献报道SLPI的分子量相符。
本研究首次利用毕赤酵母成功表达SLPI蛋白,为基因工程开发hSLPI重组药物奠定理论和实验基础。
SLPI(secretory leukocyte peptidase inhibitor)is one of the two members ofthe ALP superfamily of proteinase inhibitors and a kind of whey acidic protein(WAP) motif protein. The mature SLPI consists of 107 amino acids, which iscleavaged from 14.3kDa SLPI precursor with a signal peptide of 25 amino acids.SLPI is a non-glycosylated, hydrophobic, highly basic (pI >. 9.5), acid-stable,cationic 11.7-kDa single-chain polypeptide serine protease inhibitor, relativelyheat-stable, consisting of two homologous cystein-rich domains of 53 and 54amino acids. The human SLPI gene is localized on chromosome 20q12-13.2.The SLPI gene consists of four exons and three introns and spans approximately2.6 kb, which can be modulated at both the transcriptional and translationallevels. To date, no polymorphism of the SLPI gene and no state of SLPIdeficiency have been found. The SLPI gene belongs to the trappin gene family.The products of this family are characterized by an N-terminal transglutaminasedomain substrate and a C-terminal four-disulfide core. These two domains(COOH terminal and NH2 terminal) share about 35% homology. Each of thesedomains has distinct enzyme activities. The tertiary structure of the SLPImolecule resembles a boomerang, with each arm carrying one domain. Thefour-in-each-domain disulfide bridges formed between the 16 cysteine residues,as well as the two-domain interaction, thereby connecting the polypeptide
segments of the molecule, contribute to the conformation and efficacy of themolecule.SLPI is a potent inhibitor of a variety of endogenous proteolytic enzymes andcould be in host defence against invading micro-organisms, and even the DNAduplication of virus. Along with the more and more understanding of thisprotein, people have realized that it is an innate immune reaction for SLPI toprotect local mucus membrane and tissue against the inflammatory injuries. It isgenerally postulated that the balance between proteinases and antiproteinases isa prerequisite for the maintenance of tissue integrity. Indeed, it is showed thatcleavage of SLPI results in increased tissue damage. The protease inhibitoryactivity is restricted to the COOH-terminal domain with the active center beingformed by the [Leu.sup.72]-[Met.sup.73] residues. The NH2-terminal domain(N-terminal domain) has no such properties, but it may aid in stabilizing theprotease-antiprotease complex and may mediate the enhancement of theantiproteinase activity of SLPI by heparin. Heparin augments the effectivenessof SLPI as it induces a conformational change in the inhibitor. It seems that theN-terminal domain is responsible for the dose-dependent bactericidal propertiesof SLPI against both gram-positive (S. aureus) and gram-negative (E. coli)bacteria.Recent scientific evidence suggests that SLPI has broad-spectrumantibiotic activity that includes bactericidal and antifungal properties. As withthe antibacterial-bactericidal activity, the antifungal activity was mainlylocalized in the NH2-terminal domain. It is believed that killing of fungusprotects the epithelia from the fungal proteases. Probably the antibacterial andantifungal activities are related to the cationic nature of SLPI.
SLPI distribution in vivo is mucosa-associated.It is synthesized and secretedin several glandular organs including the lung, mainly expressed in respiratorytract and germinal epithelium and is present constitutively in most mucosalsecretion (hence its alternative name mucus proteinase inhibitor). SLPI is foundin various secretory fluids including parotid secretions, bronchial, nasal, andcervical mucous, and seminal fluid. SLPI was originally isolated from parotidsaliva and has been detected in a variety secretions such as pancreatic secretion,whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears,and cerebral spinal fluid, as in secretions from the nose and bronchi, etc. SLPI isan important component of the antiprotease defense of tissues bathed bysecretory fluids and could be useful as a therapeutic agent for treatment of theproteolytic damage of tissues seen in degenerative and inflammatory diseases.SLPI protects the tissues by inhibiting the proteases, such as cathepsin G,elastase, and trypsin from neutrophils;chymotrypsin and trypsin from pancreaticacinar cells;and chymase and tryptase from mast cells. The SLPI gene wasfound to be expressed in lung, breast, oropharyngeal, bladder, endometrial,ovarian, and colorectal carcinomas, and SLPI detection is correlated with poorprognosis. SLPI is also found in neurons and astrocytes in the ischemic braintissue. Finally, SLPI was found to play a pivotal role in apoptosis and woundhealing.To date, hSLPI has mainly been extracted from tissues/cells or expressed inE.coli through gene engineering. But these methods have some insurmountableflaws,which confine the application of SLPI on a large scale. The origin oftissues/cells for protein extraction is limited and it is difficult in purification, and
prone to cross contamination. As a host E.coli. has shortcomings such asendotoxin generating, plasmid liable to be lost, and so on, which make it neversurpass eukaryotic expression system. The foreign gene expression in E.coli. isinfluenced a lot. The cationic property of SLPI may bind to mRNA and DNA asa possible cause of toxicity to E.coli., which makes it impossible to be expressedat high level in E.coli. Adenovirus mediated gene therapy also has potentialdeficiency:(1) lack of target-specific action;(2) unable to be integrated into thehost DNA, leading to the short term of expression;(3) intense immune reactionand cell toxicity, which may be lethal to the host.According to the mentioned problems,we chose the Pichia pastoris to expressrecombinant protein. Pichia pastoris is unicellular eukaryotic organism, and it iscommonly used for expression recombinant protein in recent years. The benefitsof expression recombinant protein base on the following merits: the strongalcohol oxidase (AOX) promoter can control the foreign gene to express strictly;it can be cultureed with high density but require low nourishment which isbeneficial for industrial manufacture;it has very few self secrete protein and dogood for purification of foreign protein;the foreign gene is stable when it istransformed to the chromosome of the Pichia pastoris;it can process anddecorate the expression protein by itself, methylotrophic yeasts have the abilityto use methanol as a sole source of carbon and energy. Up until now expressionof SLPI in Pichia pastoris has not been found in any reports.In order to express human recombinant SLPI in Pichia pastoris we have donesuch work as follows:The construction of human recombinant SLPI expression system
1. The construction of cloning vector contains HER2/neu ECD geneSLPI cDNA was cloned from Hella cell (SLPI over-expression in humancervical carcinoma cell line HeLa) through reverse transcription polymerasechain reaction (RT-PCR 5' primer5'-TCACTGCAGCCTTCACCATGAAGTCCAG, 3' prime5'-CTCCTCGAGATGGCAGGAATCAAGCTTTC). The SLPI DNA wassubcloned into a PBS clone plasmid. The recombinant vector was identified bydigestion of restriction enzyme PstI and XhoI. The result showed that the newrecombinant SLPI cloning vector was constructed successfully. DNAsequencing results showed that the cloning vector contained the complete genesequence of SLPI.2. The construction of expression vector contained SLPI geneTaking the cloning plasmid pBS-SLPI as the template, PCR (5' primer 5'-TGACTCGAGAAGAGATCTGGAAAGTCCTTC, 3' primer 5'-CGGTCTAGATTAAGCTTTCACAGGGGAAAC) amplified the DNA of theinterest gene. We constructed an expression plasmid pPICZα comprising SLPIgene. The recombinant vector was identified by the digestion of restrictionenzyme XhoI and XbaI. The result showed that the new recombinant SLPIexpression vector was constructed successfully. DNA sequencing results showthat the expression plasmid pPICZα-SLPI contained the correct reading frame.3. Transformation of the recombinant plasmid, identification of the positiveclones and the expression of the recombinant protein.Mixed 80ul of the fresh competence of yeast cells with 5～10μg of linearizedpPICZα-SLPI (in 5～10μl sterile water) and transfered them to an ice-cold
(0℃) 0.2cm electroporation cuvette. Spread 50～100μl of each on separate, andlabeled YPD plates containing (25μg/ml) of Zeocin. To incubate plates for 2 to3 days at 28℃ until colonies formed. Positive transformants were selected andcultured in BMGY separately. When the Pichia pastoris were cultured in thelogarithmic phase , took 1ml culture liquid to extract the genome DNA of Pichiapastoris. And the SLPI transformants were assayed via PCR withno-transformant as antitheses. The result showed all transformants have theDNA fragment of 340bp but the antitheses has no DNA fragment of the interest.The Pichia pastoris in the logarithmic phase were centrifuged by1500r/minfor 5min. To resuspend the Pichia pastoris in the culture medium of BMMY andadd methanol with the concentration of 0.5%. It was ready to express of thegene of interest.The transformants were selected and induced by 0.5% methanol in BMMYmedium. The transformants were moved to orbital shaker in 28℃, with225r/min, and shaked to express.4. Analysis of the human recombinant SLPI proteinTook 20ul cultured supernatant of the 72h, which have been condensed byacetone for 2 to 12 times. The recombinant of the SLPI protein was detected bythe 15% SDS-PAGE and a protein band can be seen in the zone of 12kDa.For the first time SLPI protein was expressed in Pichia pastoris and thisprovides a basis for the developing research.
目前已可由重组DNA技术得到SLPI,但因成本高尚未广泛用于临床。从人粘膜分泌液中提取SLPI,存在原料来源有限、病毒不易灭活和纯度不高等问题。市场上重组人SLPI是以大肠杆菌为表达载体的,不但具有原核表达系统永远无法超越真核表达系统的缺陷,而且SLPI蛋白的阳离子特性能会对大肠杆菌产生毒害作用并影响其生存繁殖,所以无法实现高表达,难以满足医学研究的需要。目前尚未见有用毕赤酵母表达SLPI蛋白的研究报道。鉴于此,本研究选用毕赤酵母作为SLPI蛋白生产的生物反应器,开展以下工作:
1.本研究构建克隆载体pBS-SLPI,经测序证明序列正确,含有完整的人SLPI基因。以此为基础,可以通过重组改造实现病毒载体基因治疗,也可以进行下一步重组蛋白的研究。
2.本研究构建表达载体pPICZα-SLPI,经测序证实含有SLPI正确读框结构。用毕赤酵母表达SLPI蛋白经15% SDS-PAGE电泳分析,在蛋白质分子量12kDa附近可见一条考马斯亮蓝染色蛋白区带,与文献报道SLPI的分子量相符。
本研究首次利用毕赤酵母成功表达SLPI蛋白,为基因工程开发hSLPI重组药物奠定理论和实验基础。
SLPI(secretory leukocyte peptidase inhibitor)is one of the two members ofthe ALP superfamily of proteinase inhibitors and a kind of whey acidic protein(WAP) motif protein. The mature SLPI consists of 107 amino acids, which iscleavaged from 14.3kDa SLPI precursor with a signal peptide of 25 amino acids.SLPI is a non-glycosylated, hydrophobic, highly basic (pI >. 9.5), acid-stable,cationic 11.7-kDa single-chain polypeptide serine protease inhibitor, relativelyheat-stable, consisting of two homologous cystein-rich domains of 53 and 54amino acids. The human SLPI gene is localized on chromosome 20q12-13.2.The SLPI gene consists of four exons and three introns and spans approximately2.6 kb, which can be modulated at both the transcriptional and translationallevels. To date, no polymorphism of the SLPI gene and no state of SLPIdeficiency have been found. The SLPI gene belongs to the trappin gene family.The products of this family are characterized by an N-terminal transglutaminasedomain substrate and a C-terminal four-disulfide core. These two domains(COOH terminal and NH2 terminal) share about 35% homology. Each of thesedomains has distinct enzyme activities. The tertiary structure of the SLPImolecule resembles a boomerang, with each arm carrying one domain. Thefour-in-each-domain disulfide bridges formed between the 16 cysteine residues,as well as the two-domain interaction, thereby connecting the polypeptide
segments of the molecule, contribute to the conformation and efficacy of themolecule.SLPI is a potent inhibitor of a variety of endogenous proteolytic enzymes andcould be in host defence against invading micro-organisms, and even the DNAduplication of virus. Along with the more and more understanding of thisprotein, people have realized that it is an innate immune reaction for SLPI toprotect local mucus membrane and tissue against the inflammatory injuries. It isgenerally postulated that the balance between proteinases and antiproteinases isa prerequisite for the maintenance of tissue integrity. Indeed, it is showed thatcleavage of SLPI results in increased tissue damage. The protease inhibitoryactivity is restricted to the COOH-terminal domain with the active center beingformed by the [Leu.sup.72]-[Met.sup.73] residues. The NH2-terminal domain(N-terminal domain) has no such properties, but it may aid in stabilizing theprotease-antiprotease complex and may mediate the enhancement of theantiproteinase activity of SLPI by heparin. Heparin augments the effectivenessof SLPI as it induces a conformational change in the inhibitor. It seems that theN-terminal domain is responsible for the dose-dependent bactericidal propertiesof SLPI against both gram-positive (S. aureus) and gram-negative (E. coli)bacteria.Recent scientific evidence suggests that SLPI has broad-spectrumantibiotic activity that includes bactericidal and antifungal properties. As withthe antibacterial-bactericidal activity, the antifungal activity was mainlylocalized in the NH2-terminal domain. It is believed that killing of fungusprotects the epithelia from the fungal proteases. Probably the antibacterial andantifungal activities are related to the cationic nature of SLPI.
SLPI distribution in vivo is mucosa-associated.It is synthesized and secretedin several glandular organs including the lung, mainly expressed in respiratorytract and germinal epithelium and is present constitutively in most mucosalsecretion (hence its alternative name mucus proteinase inhibitor). SLPI is foundin various secretory fluids including parotid secretions, bronchial, nasal, andcervical mucous, and seminal fluid. SLPI was originally isolated from parotidsaliva and has been detected in a variety secretions such as pancreatic secretion,whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears,and cerebral spinal fluid, as in secretions from the nose and bronchi, etc. SLPI isan important component of the antiprotease defense of tissues bathed bysecretory fluids and could be useful as a therapeutic agent for treatment of theproteolytic damage of tissues seen in degenerative and inflammatory diseases.SLPI protects the tissues by inhibiting the proteases, such as cathepsin G,elastase, and trypsin from neutrophils;chymotrypsin and trypsin from pancreaticacinar cells;and chymase and tryptase from mast cells. The SLPI gene wasfound to be expressed in lung, breast, oropharyngeal, bladder, endometrial,ovarian, and colorectal carcinomas, and SLPI detection is correlated with poorprognosis. SLPI is also found in neurons and astrocytes in the ischemic braintissue. Finally, SLPI was found to play a pivotal role in apoptosis and woundhealing.To date, hSLPI has mainly been extracted from tissues/cells or expressed inE.coli through gene engineering. But these methods have some insurmountableflaws,which confine the application of SLPI on a large scale. The origin oftissues/cells for protein extraction is limited and it is difficult in purification, and
prone to cross contamination. As a host E.coli. has shortcomings such asendotoxin generating, plasmid liable to be lost, and so on, which make it neversurpass eukaryotic expression system. The foreign gene expression in E.coli. isinfluenced a lot. The cationic property of SLPI may bind to mRNA and DNA asa possible cause of toxicity to E.coli., which makes it impossible to be expressedat high level in E.coli. Adenovirus mediated gene therapy also has potentialdeficiency:(1) lack of target-specific action;(2) unable to be integrated into thehost DNA, leading to the short term of expression;(3) intense immune reactionand cell toxicity, which may be lethal to the host.According to the mentioned problems,we chose the Pichia pastoris to expressrecombinant protein. Pichia pastoris is unicellular eukaryotic organism, and it iscommonly used for expression recombinant protein in recent years. The benefitsof expression recombinant protein base on the following merits: the strongalcohol oxidase (AOX) promoter can control the foreign gene to express strictly;it can be cultureed with high density but require low nourishment which isbeneficial for industrial manufacture;it has very few self secrete protein and dogood for purification of foreign protein;the foreign gene is stable when it istransformed to the chromosome of the Pichia pastoris;it can process anddecorate the expression protein by itself, methylotrophic yeasts have the abilityto use methanol as a sole source of carbon and energy. Up until now expressionof SLPI in Pichia pastoris has not been found in any reports.In order to express human recombinant SLPI in Pichia pastoris we have donesuch work as follows:The construction of human recombinant SLPI expression system
1. The construction of cloning vector contains HER2/neu ECD geneSLPI cDNA was cloned from Hella cell (SLPI over-expression in humancervical carcinoma cell line HeLa) through reverse transcription polymerasechain reaction (RT-PCR 5' primer5'-TCACTGCAGCCTTCACCATGAAGTCCAG, 3' prime5'-CTCCTCGAGATGGCAGGAATCAAGCTTTC). The SLPI DNA wassubcloned into a PBS clone plasmid. The recombinant vector was identified bydigestion of restriction enzyme PstI and XhoI. The result showed that the newrecombinant SLPI cloning vector was constructed successfully. DNAsequencing results showed that the cloning vector contained the complete genesequence of SLPI.2. The construction of expression vector contained SLPI geneTaking the cloning plasmid pBS-SLPI as the template, PCR (5' primer 5'-TGACTCGAGAAGAGATCTGGAAAGTCCTTC, 3' primer 5'-CGGTCTAGATTAAGCTTTCACAGGGGAAAC) amplified the DNA of theinterest gene. We constructed an expression plasmid pPICZα comprising SLPIgene. The recombinant vector was identified by the digestion of restrictionenzyme XhoI and XbaI. The result showed that the new recombinant SLPIexpression vector was constructed successfully. DNA sequencing results showthat the expression plasmid pPICZα-SLPI contained the correct reading frame.3. Transformation of the recombinant plasmid, identification of the positiveclones and the expression of the recombinant protein.Mixed 80ul of the fresh competence of yeast cells with 5～10μg of linearizedpPICZα-SLPI (in 5～10μl sterile water) and transfered them to an ice-cold
(0℃) 0.2cm electroporation cuvette. Spread 50～100μl of each on separate, andlabeled YPD plates containing (25μg/ml) of Zeocin. To incubate plates for 2 to3 days at 28℃ until colonies formed. Positive transformants were selected andcultured in BMGY separately. When the Pichia pastoris were cultured in thelogarithmic phase , took 1ml culture liquid to extract the genome DNA of Pichiapastoris. And the SLPI transformants were assayed via PCR withno-transformant as antitheses. The result showed all transformants have theDNA fragment of 340bp but the antitheses has no DNA fragment of the interest.The Pichia pastoris in the logarithmic phase were centrifuged by1500r/minfor 5min. To resuspend the Pichia pastoris in the culture medium of BMMY andadd methanol with the concentration of 0.5%. It was ready to express of thegene of interest.The transformants were selected and induced by 0.5% methanol in BMMYmedium. The transformants were moved to orbital shaker in 28℃, with225r/min, and shaked to express.4. Analysis of the human recombinant SLPI proteinTook 20ul cultured supernatant of the 72h, which have been condensed byacetone for 2 to 12 times. The recombinant of the SLPI protein was detected bythe 15% SDS-PAGE and a protein band can be seen in the zone of 12kDa.For the first time SLPI protein was expressed in Pichia pastoris and thisprovides a basis for the developing research.
引文
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36. Odaka, C., T. Mizuochi, J. Yang, and A. Ding. Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response. J. Immunol.2003;171:1507-1514.
37. Sorensen, O. E., J. B. Cowland, K. Theilgaard-Monch, L. Liu, T. Ganz, and N. Borregaard. Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes, a consequence of common growth factors. J. Immunol. 2003;170:5583-5589.
38. Ogra, P. I., J. Mestecky, and M. E. Lamm. Proteinase inhibitors, 1999;p. 97. In P. I. Ogra, J. Mestecky, and M. E. Lamm (ed.), Mucosal immunology, 2nd ed. Academic Press, San Diego, Calif.
39. Zhu J, Nathan C, Jin W, Sin D, Ashcroft GS, Wahl SM, Lacmis L, Erdjument-Bromage H, Tempst P, Wright CD, Ding A. Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell. 2002;Dec 13;111(6):867-78
40. Wright CD, Kennedy JA, Zitnik RJ, Kashem MA. Inhibition of murine neutrophil serine proteinases by human and murine secretory leukocyte protease inhibitor. Biochem Biophys Res Commun. 1999;Jan 27;254(3):614-7.
41. Saitoh H, Masuda T, Shimura S, Fushimi T, Shirato K. Secrition and gene expression of secretory leukocyte protease inhibitor by human airway submucosal glands. Am J Physiol Lung Cell Mol Physiol.2001;Jan;289(1):L79-87.
42. Masuda K, Kamimura T, Kanesaki M, et al. Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombincleavable fusion protein in Escherichia coli. Protein Eng, 1996, 9: 101-106.
43. Hagiwara K, Kikuchi T, Endo Y, Huqun, Usui K, Takahashi M, Shibata N, Kusakabe T, Xin H, Hoshi S, Miki M, Inooka N, Tokue Y, Nukiwa I. Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single why acidic protein motif. J Immunol.2003;Feb 15;170(4):1973-9.
44. Schalkwijk J, Wiedow O, Hirose S: The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core.Biochem J 1999, 340:569-577.
45. Simpson AJ, Maxwell AI, Govan JRW, Haslett C, Sallenave JM: Elafin (elastase-specific inhibitor) has anti-microbial activity against Gram-positive and Gram-negative respiratory pathogens.FEBS Lett 1999, 452:309-313.
46. Jin FY, Nathan CF, Radzioch D, Ding A: Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor.Infect Immun 1998,66:2447-2452.
47. Zhang Y, DeWitt DL, McNeely TB, Wahl SM, Wahl LM: Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. J Clin Invest 1997, 199:894-900.
48. McElvancy NG,Doujaiji B,Moan MJ,Burnham MR,Wu MC,Crystal RG.Pharmacokinetics of recombinant secretory leukoprotease inhibitor aerosolized to normals and individuals with cystic fibrosis. Am Rev Respir Dis.1993 Oct;148(4 Pt 1):1056-60.
49. Mackiewicz, I., Kushner, I. and Baumann, H. (eds.) (1993) Acute Phase Proteins. Molecular Biology, Biochemistry, and Clinical Applications, CRC Press, Boca Raton, FL
50. Kramps JA, Franken C, Dijkman JH. ELISA for quantitative measurement of low-molecular-weight bronchial protease inhibitor in human sputum. Am Rev Respir Dis.1984 Jun;129(6):959-63.
51. Pfundt R,van Ruissen F,van Vlijmen-Willems IM,Alkemade HA,Zeeuwen PL,Jap PH,Dijkman H,Fransen J,Croes H,van ErpPE,Schalkwijk J. Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia. Clin Invest.1996 Sep;15;98(6):13899-99.
52. Nara K,Ito S,Ito T,Suzuki Y,Ghoneim MA,Tachibana S,Hirose S.Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed “cementoin”.J Biochem(Tokyo).1994 Mar;115(3):441-8.
53. Perlmutter DH,May LT,Sehgal PB. Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.J Clin Invest.1989 Jul;84(1):138-44.
54. da Silva MC , Zahm JM, Gras D , et al . Dynamic interaction between airway epithelial cells and Staphylococcus aureus . Am J Physiol Lung Cell Mol Physiol , 2004;287 (3) : 5432551.
55. Nathan C. Points of control in inflammation. Nature , 2002 , 420 : 8462 852.
56. Gipson, T. S., N. M. Bless, and T. P. Shanley. Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury. J. Immunol. 1999.162:3653-3662.
57. He, S. H., P. Chen, and H. Q. Chen. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors. Acta Pharm. Sin. 2003.24:923-929.
58. Smith, C. E., and D. A. Johnson. Human bronchial leucocyte proteinase inhibitor. Biochem. J. 1985. 225:463-472.
59. Travis, J., and H. Fritz. Potential problems in designing elastase inhibitors for therapy. Am. Rev. Respir. Dis. 1991.143:1412-1415.
60. Hiemstra, P. S. Novel roles of protease inhibitors in infection and inflammation. Biochem. Soc. Trans. 2000.30:116-120.
61. Tomee, J. F. C., G. H. Kolter, P. S. Hiemstra, and H. F. Kauffman. Secretory leucoprotease inhibitor: a native antimicrobial protein presenting a new therapeutic opinion? Thorax 1998. 53:114-116.
62. Eisenberg, S. P., K. K. Hale, P. Heimdal, and R. C. Thompson. Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. J. Biol. Chem. 1990. 265:7976-7981.
63. Van-Seuringen, I., and M. Davril. Separation of the two domains of human mucus proteinase inhibitor: inhibitory activity is only located in the carboxy-terminal domain. Biochem. Biophys. Res. Commun. 1991. 179:1587-1592.
64. Grutter, M. G., G. Frendrich, R. Huber, and W. Bode. The 2.5 ? X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymothrypsin. EMBO J. 1988. 7:345-351.
65. Ying, Q. L., M. Kemme, and S. R. Simon. Functions of the N-terminal domain of secretory leukoprotease inhibitor. Biochemistry. 1994. 33:5445-5450.
66. Marone, G., L. M. Lichtenstein, and S. J. Galli. Regulation by physiological inhibitors, In G. Marone, L. M. Lichtenstein, and S. J. Galli (ed.), Mast cells and basophils. Academic Press, San Diego, Calif. 2000. p. 278-279.
67. Greene, C., C. Taggart, and G. Lowe. Local impairment of anti-neutrophil elastase capacity in community-acquired pneumonia. J. Infect. Dis. 2003. 188:769-776.
68. Zang, Y., D. L. De Witt, T. B. McNeely, S. M. Wahl, and L. M. Wahl. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteases. J. Clin. Investig. 1997. 99:894-900.
69. Kramps, J. A., C. Franken, and J. H. Dijkman. Quantity of anti-leucoprotease relative to a1-proteinase inhibitor in peripheral airspaces of human lung. Clin. Sci. 1988.75:351-353.
70. Ding, A., N. Thieblemont, and J. Zhu. Secretory leukocyte protease inhibitor interferes with uptake of lipopolysaccharide by macrophages. Infect. Immun. 1999. 67:4485-4489.
71. Nakamura, A., Y. Mori, K. Hagiwara, and T. Suzuki. Increased susceptibility to LPS-induced endotoxin shock in secretory leukoprotease inhibitor (SLPI)-deficient mice. J. Exp. Med. 2003. 197:669-674.
72. Sano, C., T. Shimizu, and H. Tomioka. Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-kΒ activation of lipopolysaccharide-stimulated macrophages. Cytokine 2003. 21:38-42.
73. Hiemstra, P. S., R. J. Maassen, and J. Stolk. Antibacterial activity of antileukoprotease. Infect. Immun. 1996. 64:4520-4524.
74. Miller, K. W., R. J. Evans, S. P. Eisenberg, and R. C. Thompson. Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli. J. Bacteriol. 1989. 171:2166-2172.
75. Tomee, J. F. C., P. S. Hiemstra, R. Heinzel-Wieland, and H. F. Kauffman. Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi. J. Infect. Dis. 1997.176:740-747.
76. Chattopadhyay, A., L. R. Gray, L. L. Patton, and D. J. Caplan. Salivary secretory protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons. Infect. Immun. 2004. 72:1956-1963.
77. Shugars, D. C., C. A. Watkins, and H. J. Cowen. Salivary concentration of secretory leukocyte protease inhibitor, an antimicrobial protein, is decreased with advanced age. Gerontology 2001.47:246-253.
78. Challacombe, S. J., and S. P. Sweet. Oral mucosal immunity and HIV infection: current status. Oral Dis. 2002. 8(Suppl. 2):S55-S62.
79. McElvancy NG, Doujaiji B, Moan MJ, Burnham MR, Wu MC, Crystal RG.Pharmacokinetics of recombinant secretory leukoprotease inhibitor aerosolized to normals and individuals with cystic fibrosis. Am Rev Respir Dis.1993 Oct;148(4 Pt 1):1056-60.
80. Marlene Cimons and Thomas H. Maugh II. New Drugs Offer Hope in Battle Against AIDS;Health: Preliminary trials of protease inhibitors show that they are 10 to 20 times as powerful as AZT. The Times Mirror Company;Los Angeles Times -February 01, 1995, Wednesday, Home Edition PAGE: A-1.
81. Barker SD, Coolidge CJ, Kanerva A, et al. The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy. J Gene Med, 2003,5(4):300-310.
82. 杜晓媛,张桂珍,杜珍武等,人SLPI启动子调控下eGFP基因表达载体的构建与表达,中国实验诊断学,10卷2期
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3. Camilla Hollander, Max Nystr?m, abina Janciauskiene, and Ulla Westin.Human mast cells decrease SLPI levels in type II – like alveolar cell model, in vitro. Cancer Cell International 2003,3:14 doi:10.1186/1475-2867-3-14.
4. Stergios Doumas, Alexandros Kolokotronis, and Panagiotis Stefanopoulos.Anti-Inflammatory and Antimicrobial Roles of Secretory Leukocyte Protease Inhibitor. Infect Immun. 2005 March;73(3):1271–1274.doi:10.1128/IAI. 73.3.1271-1274.2005.
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9. Zhu J, Nathan C, Ding A. Suppression of macrophage responses to bacterial lipopolysaccharide by a non-secretory form of secretory leukocyte protease inhibitor. Biochim Biophys Acta. 1999;1451:219-223.
10. M G Grütter, G Fendrich, R Huber, and W Bode.The 2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymotrypsin.The EMBO Journal vol.7 no.2 pp.345-351, 1988.
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14. Thompson, R. C., and K. Ohlsson. Isolation, properties, and complete amino acid sequence of human secretory leucocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc. Natl. Acad. Sci. USA.1988.
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15. Anne E.King,Hilary O.D.Critchley and Rodney W.Kelly.Presence of secretory leukocyte protease inhibitor in human endometrium and first trimester deciduas suggests an antibacterial protective role.Molecular Human Reproductive,2000 vol.6 no.2 pp. 191-196.
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17. Sallenave, J.-M., Si-Tahar, M., Cox, G., Chignard, M. and Gauldie, Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils. J Leukoc Biol. 1997 Jun;61(6):695-702.
18. McNeely, T. B., M. Dealy, and D. J. Dripps. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro. J. Clin. Investig. 1995.96:456-464.
19. Shugars, D. C. Endogenous mucosal antiviral factors of the oral cavity.J. Infect. Dis. 1999;179(Suppl. 3):s431-s435.
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22. Farquhar, C., T. C. VanCott, D. A. C. Mbori-Ngacha, L. Horani, R. K. Bosire, J. K. Kreiss, B. A. Richardson, and G. C. John-Stewart. Salivary secretory leukocyte protease inhibitor is associated with reduced transmission of human immunodeficiency virus type 1 through breast milk. J. Infect. Dis.2002;186:1173-1176.
23. Franken, C., C. J. L. M. Meijer, and J. H. Dijkman. Tissue distribution of antileukoprotease and lysozyme in humans. J. Histochem. Cytochem.1989;37:493-498.
24. McNeely, T. B., D. C. Shugars, M. Rosendahl, C. Tucker, S. P. Eisenberg, and S. M. Wahl. Inhibition of human immunodeficiency virus type 1 infectivity by secretory leucocyte protease inhibitor occurs prior to viral reverse transcription. Blood 1997;90:1141-1149.
25. Pillay, K., A. Coutsoudis, and A. K. Agadzi-Naqvi. Secretory leukocyte protease inhibitor in vaginal fluids and perinatal human immunodeficiency virus type 1 transmission. J. Infect. Dis. 2001;183:653-656.
26. Shugars, D. C. Endogenous mucosal antiviral factors of the oral cavity. J. Infect. Dis. 1999;179(Suppl. 3):s431-s435.
27. Abe, T., N. Kobayasi, and K. Yoshimura. Expression of the secretory leukoprotease inhibitor gene in epithelial cells. J. Clin. Investig. 87:2207-2215.
28. Fahey, J. V., and C. R. Wira. 2002. Effect of menstrual status on antibacterial activity and secretory leukocyte protease inhibitor production by human uterine epithelial cells in culture. J. Infect. Dis. 2002;185:1606-1613.
29. Jin, F., C. Nathan, D. Radzioch, and A. Ding. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997;88:417-426.
30. Nystrom, M., M. Bergenfeldt, I. Ljungcrantz, A. Lindeheim, and K. Ohlsson. Production of secretory leukocyte proteinase inhibitor (SLPI) in human pancreatic beta-cells. Mediat. Inflamm. 1999;8:147-151.
31. Ohlsson, S., I. Ljungkrantz, K. Ohlsson, M. Seyelmark, and J. Wieslander. Novel distribution of the secretory leucocyte proteinase inhibitor in kidney. Mediat. Inflamm. 2001;10:347-350.
32. Garver, R. I., K. T. Goldsmith, and B. Rodu. Strategy for achieving selective killing of carcinomas. Gene Ther. 1994;1:46-50.
33. Westin, U., M. Nystr?m, I. Ljungcrantz, B. Eriksson, and K. Ohlsson. The presence of elafin, SLPI, IL1-RA and STNFα in head and neck squamous cell carcinomas and their relation to the degree of tumor differentiation. Mediat. Inflamm.2002;11:7-12.
34. Wang, X., X. Li, and L. Xu. Up-regulation of secretory leukocyte protease inhibitor (SLPI) in the brain after ischemic stroke: adenoviral expression of SLPI protects brain from ischemic injury. Mol. Pharmacol. 2003;64:833-840.
35. Ashcroft, G. S., K. Lei, and W. Jin. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nat. Med. 2000;6:1147-1153.
36. Odaka, C., T. Mizuochi, J. Yang, and A. Ding. Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response. J. Immunol.2003;171:1507-1514.
37. Sorensen, O. E., J. B. Cowland, K. Theilgaard-Monch, L. Liu, T. Ganz, and N. Borregaard. Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes, a consequence of common growth factors. J. Immunol. 2003;170:5583-5589.
38. Ogra, P. I., J. Mestecky, and M. E. Lamm. Proteinase inhibitors, 1999;p. 97. In P. I. Ogra, J. Mestecky, and M. E. Lamm (ed.), Mucosal immunology, 2nd ed. Academic Press, San Diego, Calif.
39. Zhu J, Nathan C, Jin W, Sin D, Ashcroft GS, Wahl SM, Lacmis L, Erdjument-Bromage H, Tempst P, Wright CD, Ding A. Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell. 2002;Dec 13;111(6):867-78
40. Wright CD, Kennedy JA, Zitnik RJ, Kashem MA. Inhibition of murine neutrophil serine proteinases by human and murine secretory leukocyte protease inhibitor. Biochem Biophys Res Commun. 1999;Jan 27;254(3):614-7.
41. Saitoh H, Masuda T, Shimura S, Fushimi T, Shirato K. Secrition and gene expression of secretory leukocyte protease inhibitor by human airway submucosal glands. Am J Physiol Lung Cell Mol Physiol.2001;Jan;289(1):L79-87.
42. Masuda K, Kamimura T, Kanesaki M, et al. Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombincleavable fusion protein in Escherichia coli. Protein Eng, 1996, 9: 101-106.
43. Hagiwara K, Kikuchi T, Endo Y, Huqun, Usui K, Takahashi M, Shibata N, Kusakabe T, Xin H, Hoshi S, Miki M, Inooka N, Tokue Y, Nukiwa I. Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single why acidic protein motif. J Immunol.2003;Feb 15;170(4):1973-9.
44. Schalkwijk J, Wiedow O, Hirose S: The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core.Biochem J 1999, 340:569-577.
45. Simpson AJ, Maxwell AI, Govan JRW, Haslett C, Sallenave JM: Elafin (elastase-specific inhibitor) has anti-microbial activity against Gram-positive and Gram-negative respiratory pathogens.FEBS Lett 1999, 452:309-313.
46. Jin FY, Nathan CF, Radzioch D, Ding A: Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor.Infect Immun 1998,66:2447-2452.
47. Zhang Y, DeWitt DL, McNeely TB, Wahl SM, Wahl LM: Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. J Clin Invest 1997, 199:894-900.
48. McElvancy NG,Doujaiji B,Moan MJ,Burnham MR,Wu MC,Crystal RG.Pharmacokinetics of recombinant secretory leukoprotease inhibitor aerosolized to normals and individuals with cystic fibrosis. Am Rev Respir Dis.1993 Oct;148(4 Pt 1):1056-60.
49. Mackiewicz, I., Kushner, I. and Baumann, H. (eds.) (1993) Acute Phase Proteins. Molecular Biology, Biochemistry, and Clinical Applications, CRC Press, Boca Raton, FL
50. Kramps JA, Franken C, Dijkman JH. ELISA for quantitative measurement of low-molecular-weight bronchial protease inhibitor in human sputum. Am Rev Respir Dis.1984 Jun;129(6):959-63.
51. Pfundt R,van Ruissen F,van Vlijmen-Willems IM,Alkemade HA,Zeeuwen PL,Jap PH,Dijkman H,Fransen J,Croes H,van ErpPE,Schalkwijk J. Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia. Clin Invest.1996 Sep;15;98(6):13899-99.
52. Nara K,Ito S,Ito T,Suzuki Y,Ghoneim MA,Tachibana S,Hirose S.Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed “cementoin”.J Biochem(Tokyo).1994 Mar;115(3):441-8.
53. Perlmutter DH,May LT,Sehgal PB. Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.J Clin Invest.1989 Jul;84(1):138-44.
54. da Silva MC , Zahm JM, Gras D , et al . Dynamic interaction between airway epithelial cells and Staphylococcus aureus . Am J Physiol Lung Cell Mol Physiol , 2004;287 (3) : 5432551.
55. Nathan C. Points of control in inflammation. Nature , 2002 , 420 : 8462 852.
56. Gipson, T. S., N. M. Bless, and T. P. Shanley. Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury. J. Immunol. 1999.162:3653-3662.
57. He, S. H., P. Chen, and H. Q. Chen. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors. Acta Pharm. Sin. 2003.24:923-929.
58. Smith, C. E., and D. A. Johnson. Human bronchial leucocyte proteinase inhibitor. Biochem. J. 1985. 225:463-472.
59. Travis, J., and H. Fritz. Potential problems in designing elastase inhibitors for therapy. Am. Rev. Respir. Dis. 1991.143:1412-1415.
60. Hiemstra, P. S. Novel roles of protease inhibitors in infection and inflammation. Biochem. Soc. Trans. 2000.30:116-120.
61. Tomee, J. F. C., G. H. Kolter, P. S. Hiemstra, and H. F. Kauffman. Secretory leucoprotease inhibitor: a native antimicrobial protein presenting a new therapeutic opinion? Thorax 1998. 53:114-116.
62. Eisenberg, S. P., K. K. Hale, P. Heimdal, and R. C. Thompson. Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. J. Biol. Chem. 1990. 265:7976-7981.
63. Van-Seuringen, I., and M. Davril. Separation of the two domains of human mucus proteinase inhibitor: inhibitory activity is only located in the carboxy-terminal domain. Biochem. Biophys. Res. Commun. 1991. 179:1587-1592.
64. Grutter, M. G., G. Frendrich, R. Huber, and W. Bode. The 2.5 ? X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymothrypsin. EMBO J. 1988. 7:345-351.
65. Ying, Q. L., M. Kemme, and S. R. Simon. Functions of the N-terminal domain of secretory leukoprotease inhibitor. Biochemistry. 1994. 33:5445-5450.
66. Marone, G., L. M. Lichtenstein, and S. J. Galli. Regulation by physiological inhibitors, In G. Marone, L. M. Lichtenstein, and S. J. Galli (ed.), Mast cells and basophils. Academic Press, San Diego, Calif. 2000. p. 278-279.
67. Greene, C., C. Taggart, and G. Lowe. Local impairment of anti-neutrophil elastase capacity in community-acquired pneumonia. J. Infect. Dis. 2003. 188:769-776.
68. Zang, Y., D. L. De Witt, T. B. McNeely, S. M. Wahl, and L. M. Wahl. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteases. J. Clin. Investig. 1997. 99:894-900.
69. Kramps, J. A., C. Franken, and J. H. Dijkman. Quantity of anti-leucoprotease relative to a1-proteinase inhibitor in peripheral airspaces of human lung. Clin. Sci. 1988.75:351-353.
70. Ding, A., N. Thieblemont, and J. Zhu. Secretory leukocyte protease inhibitor interferes with uptake of lipopolysaccharide by macrophages. Infect. Immun. 1999. 67:4485-4489.
71. Nakamura, A., Y. Mori, K. Hagiwara, and T. Suzuki. Increased susceptibility to LPS-induced endotoxin shock in secretory leukoprotease inhibitor (SLPI)-deficient mice. J. Exp. Med. 2003. 197:669-674.
72. Sano, C., T. Shimizu, and H. Tomioka. Effects of secretory leukocyte protease inhibitor on the tumor necrosis factor-alpha production and NF-kΒ activation of lipopolysaccharide-stimulated macrophages. Cytokine 2003. 21:38-42.
73. Hiemstra, P. S., R. J. Maassen, and J. Stolk. Antibacterial activity of antileukoprotease. Infect. Immun. 1996. 64:4520-4524.
74. Miller, K. W., R. J. Evans, S. P. Eisenberg, and R. C. Thompson. Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli. J. Bacteriol. 1989. 171:2166-2172.
75. Tomee, J. F. C., P. S. Hiemstra, R. Heinzel-Wieland, and H. F. Kauffman. Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi. J. Infect. Dis. 1997.176:740-747.
76. Chattopadhyay, A., L. R. Gray, L. L. Patton, and D. J. Caplan. Salivary secretory protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons. Infect. Immun. 2004. 72:1956-1963.
77. Shugars, D. C., C. A. Watkins, and H. J. Cowen. Salivary concentration of secretory leukocyte protease inhibitor, an antimicrobial protein, is decreased with advanced age. Gerontology 2001.47:246-253.
78. Challacombe, S. J., and S. P. Sweet. Oral mucosal immunity and HIV infection: current status. Oral Dis. 2002. 8(Suppl. 2):S55-S62.
79. McElvancy NG, Doujaiji B, Moan MJ, Burnham MR, Wu MC, Crystal RG.Pharmacokinetics of recombinant secretory leukoprotease inhibitor aerosolized to normals and individuals with cystic fibrosis. Am Rev Respir Dis.1993 Oct;148(4 Pt 1):1056-60.
80. Marlene Cimons and Thomas H. Maugh II. New Drugs Offer Hope in Battle Against AIDS;Health: Preliminary trials of protease inhibitors show that they are 10 to 20 times as powerful as AZT. The Times Mirror Company;Los Angeles Times -February 01, 1995, Wednesday, Home Edition PAGE: A-1.
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