中国林蛙基因工程抗菌肽制备及生物学特性研究
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摘要
本研究应用基因工程方法首次从中国林蛙皮肤组织中扩增了一个新的抗菌肽基因,对扩增的序列进行分析,结果表明,扩增的序列为一个新的中国林蛙抗菌肽基因;构建了此基因的原核表达质粒和真核表达质粒,经在大肠杆菌和酵母菌中诱导表达,筛选出了能够表达较高生物活性抗菌肽的酵母表达株;用纯化的表达产物进行体外抑菌谱、抑瘤作用、热稳定性及作用模式的研究。结果表明,G+和G-均表现抗性,可以体外抑杀肿瘤细胞,但不对正常人胚肾细胞无明显抑杀作用;温度和pH值对重组表达抗菌肽活性没有明显的影响;用重组表达抗菌肽的进行细胞溶血性实验、抗炎作用以及对血管通透性等小鼠体内安全性实验,结果表明,重组表达的抗菌肽有一定的溶血活性;能明显降低致炎剂导致的小鼠耳肿胀,能明显抑制小鼠血管通透性,从而降低炎症的发生率;给实验小鼠口服重组表达抗菌肽后,实验组小鼠体重一过性下降后恢复正常。本研究克隆并鉴定了一个新的中国林蛙抗菌肽基因,并比较和优化了中国林蛙抗菌肽在不同表达系统中表达、纯化等一系列的实验条件。确定了使用酵母表达系统表达重组林蛙抗菌肽的条件。利用得到的重组林蛙抗菌肽蛋白,进行生物活性研究,从而为基因工程生产重组林蛙抗菌肽和该抗菌肽的应用研究奠定了基础。
Antibiotic peptide are a group of small molecule peptides produced by plants,insects, amphibians and mammals including human beings. These peptides have manybiological activities including antibacterial, antifungal, antivirus, antitumor andantiparasite. The antibiotic peptides is encoded by an ORF. The antibiotic peptides arethe most important nature defense material of the amphibians because of their specialliving environment, which have high effective and broad-spectrum antibacterialactivities and hardly inducing drug-resistance. Because a lot of drug-resistance strainsare emerging prominently at present, the antibiotic peptides of amphibians have themany chance to be a new kind of antibacterial drug for clinical use. Extracting natureamphibians antibiotic peptides have the limits of little raw material, high cost, lowyield and complex process, the purpose of this research is to overcome these defectsand to produce the peptide by the method of gene engineering in order to be research anew path for prepate abundanceantibacterial peptide.
Cloning and sequencing of the rana skin antibiotic peptide gene. Three pairs ofdegenerate primers were designed and synthesized according to the frog skinantibacterial peptide gene reported in GenBank and the rana skin antibiotic peptidegene was amplified by RT-PCR from rana Skin. The structural gene of rana skinantibiotic peptide gene was cloned, sequenced and phylogenesis analysised, and theresult showed that, compared with the reported gene sequence in Genbank, thehomology of the complete DNA sequence was 45.8-74.2% and the deduced aminoacid sequence was 12.7-147.7%,the major variability region was localized in the
mature peptide gene sequence, and the signal peptide gene was highly conservative.According to the sequenced rana skin antibiotic peptide gene, the hydrophobicity,surface polarity, isoelectric point, molecular weight, secondary structure of theantibiotic peptide was predicted using the software DNAstar, showing that the maturepeptide was consited of 67 aa, The molecular weight was 7943.2 Dalton, The pH was4.62, the isoelectric point was 8.95,the predicted second structure was mainly α-spiral and random coil, seldomβ-folding with highly amphipathic properties. Theapparentely variation between the cloned gene and the reported gene in Genbankshowed that this a new antibiotic peptide gene.Expression of the recombinant rana skin antibacterial peptide in E.coli. The genewas amplified from the recombinant plasmid pGEM-TRC carrying the fragmentencoding rana antibacterial peptide by PCR, which was then cloned into theexpression vector pEX-3X, so that the rana antibacterial peptide gene expressionplasmid pEX-3XRC was constructed. Positive plasmid was identified by doublerestriction enzyme digestion and PCR, and then transformed into the competent celland induced with IPTG. The expressed recombinant protein was purified. The resultsshowed that the rana antibacterial peptide gene was expressed from the recombinantcell pEX-3XRC/DE3.The optimal conditions were determined that the induction timewas 4 hours, the temperature was 37℃, the pH was 7.3 and the IPTG concentrationwas 1mmol/L,under this conditions the expressed recombinant protein,existed in formof inclusion bodies,was about 3.65mg/ml,.Expression of the recombinant rana antibiotic peptide using Pichia pastorisexpression system. The Pichia pastoris expression system is being used successfullyfor the production of various recombinant heterologous proteins and becoming thefirst expression system for a series of production. In order to study the actiivites and toprovide relevant prophase test data for large production in the future, the ranaantibiotic peptide was expressed using Pichia pastoris expression system. Thestrategies for the expression of the antibiotic peptide in the expression system. Thehost strain was GS115, the secreting type vector was pPIC9K and the recombinantgene was under the control of the Pichia pastoris α-factor. The expression systemwas then consisted of complete rana skin antibiotic peptide gene , the secreting typevector pPIC9K vector and the host Pichia pastoris GS115.The expression level of
recombinant protein in the supernatant was test by PCR,SDS-PAGE and assay of theantibacterial activities in vitro, the result showed that every transformedpPIC9KPlw/GS115strain could expressed the recombinant peptide and the expressedpeptide could inhibit relevant bacterial. The expression of the rana skin antibiotic geneand the purification of the recombinant peptide. After determination of the expressionsystem and conditions, the rana skin antibiotic peptide gene was expressed in P.pastoris GS115 carrying the expression vector pPIC9K in which the completeantibiotic peptide gene was cloned. The positive clones were screened by thementhods of plate G418 pressure and His auxotroph and a total of 55 strains werescreened. The antibacterial activites of the recombinant peptides were determined bythe way of shake-flask and three of the 3 strains showed higher activites, theproducing host were named BR7,BR9 and BRw2 ,respectively. The BR9 was used asthe seed strain and the BR9, the BRw2 were used as reserved strains. The optimationof large-scale shake flask culture of the recombinant strain pPIC9KBR9/GS115. Whenthe ratio of the medium BMGY:BMMY was 1:1 and even enlarge the total volum ,The result of the optimation of the methanol concentration was 1%, pH and OD valuewere pH 5.0 and OD600 2.0 before induction. In order to keep the recombinant proteinfolding correctly, keep the pH value 7.0 and culture 28℃ after inducing. All thesemade good foundation for high density fermentation in the future. the purification ofthe recombinant peptide. Compared the followed two purification methods anddetermined the best purification protocol: the first method was ammonium sulfateprecipitation followed by dialysis, the yield after precipitation was 10%～20% andafter dialysis was 5%;the second method was ultrafiltration and the yield was 17%.The antibacterial activity of the recombinant peptide, in vitro. The antibacterialspectra and titer of the recombinant peptide were tested by agar diffusion method, andheat and pH stability were also tested. The result showed that the antibacterial spectraof the recombinant peptide was very wide, it could inhibit some Gram-positive andGram-negative bacteria including salmonella ( S.C500 ) , Escherichia coli(ATCC25922), Bacillus subtilis (ADB403), Staphylococcus aureus (ATCC25923),streptococcus (ATCC55121), listeria (ATCC54004), Pseudomonas aeruginosa(ATCC25922), Bacillus cereus (CMCC(B)63301), Bacillus anthracis ( C40-205strain),Paeteurella(clinical isolate, swine), Klebsiella pneumoniae(clinical isolate,
Homo sapien),but could not inhibit fungus such as Candida albicans (ATCC10231))and E.coli. standard strain(ATCC25922)and engineering strain BL21(DE3).Therecombinant peptide could show its activity when temperature was between 20～65℃ and pH was 5～9, and could not lost its activity even boiled 5～10 min at 100℃.The antibacterial activity unit of the purified recombinant antibiotic peptide wasequal to 0.8×10-3ug/ml of ampicillin according to the standard of the antibacterialactivity of ampicillin.Effect on cell permeability and antibacterical activity in vivo. The activitesshowed by the purified recombinant peptide in vivo were tested in mice. Thehaemocytolysis action was tested by measuring yan-methemoglobin in the blood;anti-inflammatory action was tested by swelling of ear and effect on capillarypermeability;and effect on the growth of the mice was tested by comparing the weightbefore and after administration. The results showed that the recombinant peptide hadstrong haemolysis activities but had the activity of anti-inflammatory which couldreduce the swelling of the mice ears and inhibit vasopermeability, comparing the miceweight of the sample group with the control group before and after administrationshowed that the sample group lost 11.95%.In this study, the rana skin antibiotic peptide gene was cloned, sequenced andexpressed in E.coli and P. pastoris, respectively. All these provided data for highdensity fermentation of rana skin antibiotic peptide using P. pastoris expressionsystem and made good foundation for research of mechanism and action of thisantibiotic peptide in the future.
Antibiotic peptide are a group of small molecule peptides produced by plants,insects, amphibians and mammals including human beings. These peptides have manybiological activities including antibacterial, antifungal, antivirus, antitumor andantiparasite. The antibiotic peptides is encoded by an ORF. The antibiotic peptides arethe most important nature defense material of the amphibians because of their specialliving environment, which have high effective and broad-spectrum antibacterialactivities and hardly inducing drug-resistance. Because a lot of drug-resistance strainsare emerging prominently at present, the antibiotic peptides of amphibians have themany chance to be a new kind of antibacterial drug for clinical use. Extracting natureamphibians antibiotic peptides have the limits of little raw material, high cost, lowyield and complex process, the purpose of this research is to overcome these defectsand to produce the peptide by the method of gene engineering in order to be research anew path for prepate abundanceantibacterial peptide.
Cloning and sequencing of the rana skin antibiotic peptide gene. Three pairs ofdegenerate primers were designed and synthesized according to the frog skinantibacterial peptide gene reported in GenBank and the rana skin antibiotic peptidegene was amplified by RT-PCR from rana Skin. The structural gene of rana skinantibiotic peptide gene was cloned, sequenced and phylogenesis analysised, and theresult showed that, compared with the reported gene sequence in Genbank, thehomology of the complete DNA sequence was 45.8-74.2% and the deduced aminoacid sequence was 12.7-147.7%,the major variability region was localized in the
mature peptide gene sequence, and the signal peptide gene was highly conservative.According to the sequenced rana skin antibiotic peptide gene, the hydrophobicity,surface polarity, isoelectric point, molecular weight, secondary structure of theantibiotic peptide was predicted using the software DNAstar, showing that the maturepeptide was consited of 67 aa, The molecular weight was 7943.2 Dalton, The pH was4.62, the isoelectric point was 8.95,the predicted second structure was mainly α-spiral and random coil, seldomβ-folding with highly amphipathic properties. Theapparentely variation between the cloned gene and the reported gene in Genbankshowed that this a new antibiotic peptide gene.Expression of the recombinant rana skin antibacterial peptide in E.coli. The genewas amplified from the recombinant plasmid pGEM-TRC carrying the fragmentencoding rana antibacterial peptide by PCR, which was then cloned into theexpression vector pEX-3X, so that the rana antibacterial peptide gene expressionplasmid pEX-3XRC was constructed. Positive plasmid was identified by doublerestriction enzyme digestion and PCR, and then transformed into the competent celland induced with IPTG. The expressed recombinant protein was purified. The resultsshowed that the rana antibacterial peptide gene was expressed from the recombinantcell pEX-3XRC/DE3.The optimal conditions were determined that the induction timewas 4 hours, the temperature was 37℃, the pH was 7.3 and the IPTG concentrationwas 1mmol/L,under this conditions the expressed recombinant protein,existed in formof inclusion bodies,was about 3.65mg/ml,.Expression of the recombinant rana antibiotic peptide using Pichia pastorisexpression system. The Pichia pastoris expression system is being used successfullyfor the production of various recombinant heterologous proteins and becoming thefirst expression system for a series of production. In order to study the actiivites and toprovide relevant prophase test data for large production in the future, the ranaantibiotic peptide was expressed using Pichia pastoris expression system. Thestrategies for the expression of the antibiotic peptide in the expression system. Thehost strain was GS115, the secreting type vector was pPIC9K and the recombinantgene was under the control of the Pichia pastoris α-factor. The expression systemwas then consisted of complete rana skin antibiotic peptide gene , the secreting typevector pPIC9K vector and the host Pichia pastoris GS115.The expression level of
recombinant protein in the supernatant was test by PCR,SDS-PAGE and assay of theantibacterial activities in vitro, the result showed that every transformedpPIC9KPlw/GS115strain could expressed the recombinant peptide and the expressedpeptide could inhibit relevant bacterial. The expression of the rana skin antibiotic geneand the purification of the recombinant peptide. After determination of the expressionsystem and conditions, the rana skin antibiotic peptide gene was expressed in P.pastoris GS115 carrying the expression vector pPIC9K in which the completeantibiotic peptide gene was cloned. The positive clones were screened by thementhods of plate G418 pressure and His auxotroph and a total of 55 strains werescreened. The antibacterial activites of the recombinant peptides were determined bythe way of shake-flask and three of the 3 strains showed higher activites, theproducing host were named BR7,BR9 and BRw2 ,respectively. The BR9 was used asthe seed strain and the BR9, the BRw2 were used as reserved strains. The optimationof large-scale shake flask culture of the recombinant strain pPIC9KBR9/GS115. Whenthe ratio of the medium BMGY:BMMY was 1:1 and even enlarge the total volum ,The result of the optimation of the methanol concentration was 1%, pH and OD valuewere pH 5.0 and OD600 2.0 before induction. In order to keep the recombinant proteinfolding correctly, keep the pH value 7.0 and culture 28℃ after inducing. All thesemade good foundation for high density fermentation in the future. the purification ofthe recombinant peptide. Compared the followed two purification methods anddetermined the best purification protocol: the first method was ammonium sulfateprecipitation followed by dialysis, the yield after precipitation was 10%～20% andafter dialysis was 5%;the second method was ultrafiltration and the yield was 17%.The antibacterial activity of the recombinant peptide, in vitro. The antibacterialspectra and titer of the recombinant peptide were tested by agar diffusion method, andheat and pH stability were also tested. The result showed that the antibacterial spectraof the recombinant peptide was very wide, it could inhibit some Gram-positive andGram-negative bacteria including salmonella ( S.C500 ) , Escherichia coli(ATCC25922), Bacillus subtilis (ADB403), Staphylococcus aureus (ATCC25923),streptococcus (ATCC55121), listeria (ATCC54004), Pseudomonas aeruginosa(ATCC25922), Bacillus cereus (CMCC(B)63301), Bacillus anthracis ( C40-205strain),Paeteurella(clinical isolate, swine), Klebsiella pneumoniae(clinical isolate,
Homo sapien),but could not inhibit fungus such as Candida albicans (ATCC10231))and E.coli. standard strain(ATCC25922)and engineering strain BL21(DE3).Therecombinant peptide could show its activity when temperature was between 20～65℃ and pH was 5～9, and could not lost its activity even boiled 5～10 min at 100℃.The antibacterial activity unit of the purified recombinant antibiotic peptide wasequal to 0.8×10-3ug/ml of ampicillin according to the standard of the antibacterialactivity of ampicillin.Effect on cell permeability and antibacterical activity in vivo. The activitesshowed by the purified recombinant peptide in vivo were tested in mice. Thehaemocytolysis action was tested by measuring yan-methemoglobin in the blood;anti-inflammatory action was tested by swelling of ear and effect on capillarypermeability;and effect on the growth of the mice was tested by comparing the weightbefore and after administration. The results showed that the recombinant peptide hadstrong haemolysis activities but had the activity of anti-inflammatory which couldreduce the swelling of the mice ears and inhibit vasopermeability, comparing the miceweight of the sample group with the control group before and after administrationshowed that the sample group lost 11.95%.In this study, the rana skin antibiotic peptide gene was cloned, sequenced andexpressed in E.coli and P. pastoris, respectively. All these provided data for highdensity fermentation of rana skin antibiotic peptide using P. pastoris expressionsystem and made good foundation for research of mechanism and action of thisantibiotic peptide in the future.
引文
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