禽腺联病毒序列促进人组织激肽释放酶在鸡输卵管的表达、鉴定及检测方法建立
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摘要
腺联病毒(adeno-associated virus, AAV)是很有前景的基因治疗载体。禽腺联病毒(avian adeno-associated virus, AAAV)具有与人AAV类似的潜伏感染特性和基因组结构。为了探索AAAV末端反向重复(ITR)和Rep基因序列能否促进外源基因的整合和表达,本研究将人组织激肽释放酶(hKLK1)鸡输卵管特异表达盒克隆在AAAV ITR之间,并将CMV启动子控制的Rep基因表达盒构建于同一载体,获得重组表达载体pRep2ITR-OV4-KLK1。以不含AAAV序列的鸡输卵管表达载体pOV4-KLK1为对照,通过体外释放实验确定PEI与质粒DNA的包被条件为N/P=5;用生理盐水稀释PEI-质粒DNA复合物,经翅静脉注射产蛋鸡。蛋清中重组酶的活性检测结果显示,在质粒DNA相同的条件下, pRep2ITR-OV4-KLK1表达hK1的水平及维持时间较pOV4K有所提高或延长,但不能维持持久表达。这些试验结果提示,AAAV ITR和(或)Rep序列能促进hKLK1在鸡输卵管上皮细胞中的表达,但不能介导外源基因的整合或整合效率很低。
为了进一步研究rhKl的理化特性,对载体注射鸡蛋清进行Western blotting检测,结果显示rhK1能被hKl特异抗体识别,成熟酶的分子量为37 kDa;将鸡蛋清用不同pH的缓冲液稀释,然后进行酶活性检测,结果显示rhK1在pH7-10范围内均有较强活性;将鸡蛋清用含不同金属离子(终浓度为0.01M)的缓冲液稀释,然后进行酶活性测定,结果表明rhKl能被Cu2+、Fe3+和Al3+完全抑制;将鸡蛋清用缓冲液适当稀释,在不同温度下孵育一定时间,然后进行酶活性测定,结果显示37℃孵育20min能显著激活酶活性,而100℃孵育30min能使酶失活。
为了建立hK1的定量检测方法,以饱和硫酸铵-葡萄球菌A蛋白纯化的hKl特异单克隆抗体为包被抗体,以亲和层析纯化的GST-hKl融合蛋白为抗原,建立双抗体夹心ELISA方法。在优化的反应条件下,检测已知抗原的批内和批间重复性良好,在抗原浓度在16-125μg/ml范围内具有良好的线性关系;用建立的方法对重组载体注射鸡蛋清进行检测,结果在注射载体后的第3、6、9天,蛋清中的rhK1分别为332μg/ml、476μg/ml和248μg/ml。
Adeno-associated virus (AAV) is a promising vector for gene therapy. Avian adeno-associated virus (AAAV) has a similar persistent infection and genome structure to AAAV. To explore the feasibility of using AAAV sequences to promote oviduct-specific expression of human Human Tissue Kallikrein (rhK1) gene, we inserted the oviduct-specific expression cassette between the AAAV ITRs and included the CMV promoter-controlled Rep-expression cassette in the same vector, resulting in recombinant vector pRep2ITR-OV4-KLK1. The AAAV sequence-containing vector, as well as the control vector pOV4K, was injected into egg-laying hens via wing vein route after mixing with polyethyleneimine (PEI) at N/P=5. After twine injection, egg whites were collected for enzymatic assay. The results showed that a higher and longer hK1 expression was observed in egg whites of pRep2ITR-OV4-KLK1-injected hens than that in the control vector-injected birds. These data indicate that AAAV ITRs and/or Rep gene can promote KLK1 gene expression in an oviduct-specific manner without evidence for persistent expression.
To characterize the rhK1, egg white of the vector-injected laying hens submitted to Western blotting analysis using an antibody specific for human tissue kallikrein. The results showed that a specific protein band of 37kDa was detected, which corresponded to the mature enzyme. Further biochemical studies showed that the rhK1 had a similar thermo-stability, optimal pH and sensitivity to different ions to the natural enzymes from human and porcine tissues.
To establish a quantitative detection method for rhK1, by using affinity-purifed monoclonal antibody against hK1 as the coating antibody and the E.coli-expressed GST-KLK1 fusion protein as the known antigen, a sandwich ELISA was established. Under the optimized conditions, the ELISA reading values had intra- and inter-plate linear relationship with the antigen concentrations ranging from 16μg/ml to 125μg/ml. The egg whites of the vector–injected hens were submitted to the ELISA detection and the hK1 of 332, 476 and 248μg/ml was detected at days 3, 6 and 9 after vector injection, respectively.
为了进一步研究rhKl的理化特性,对载体注射鸡蛋清进行Western blotting检测,结果显示rhK1能被hKl特异抗体识别,成熟酶的分子量为37 kDa;将鸡蛋清用不同pH的缓冲液稀释,然后进行酶活性检测,结果显示rhK1在pH7-10范围内均有较强活性;将鸡蛋清用含不同金属离子(终浓度为0.01M)的缓冲液稀释,然后进行酶活性测定,结果表明rhKl能被Cu2+、Fe3+和Al3+完全抑制;将鸡蛋清用缓冲液适当稀释,在不同温度下孵育一定时间,然后进行酶活性测定,结果显示37℃孵育20min能显著激活酶活性,而100℃孵育30min能使酶失活。
为了建立hK1的定量检测方法,以饱和硫酸铵-葡萄球菌A蛋白纯化的hKl特异单克隆抗体为包被抗体,以亲和层析纯化的GST-hKl融合蛋白为抗原,建立双抗体夹心ELISA方法。在优化的反应条件下,检测已知抗原的批内和批间重复性良好,在抗原浓度在16-125μg/ml范围内具有良好的线性关系;用建立的方法对重组载体注射鸡蛋清进行检测,结果在注射载体后的第3、6、9天,蛋清中的rhK1分别为332μg/ml、476μg/ml和248μg/ml。
Adeno-associated virus (AAV) is a promising vector for gene therapy. Avian adeno-associated virus (AAAV) has a similar persistent infection and genome structure to AAAV. To explore the feasibility of using AAAV sequences to promote oviduct-specific expression of human Human Tissue Kallikrein (rhK1) gene, we inserted the oviduct-specific expression cassette between the AAAV ITRs and included the CMV promoter-controlled Rep-expression cassette in the same vector, resulting in recombinant vector pRep2ITR-OV4-KLK1. The AAAV sequence-containing vector, as well as the control vector pOV4K, was injected into egg-laying hens via wing vein route after mixing with polyethyleneimine (PEI) at N/P=5. After twine injection, egg whites were collected for enzymatic assay. The results showed that a higher and longer hK1 expression was observed in egg whites of pRep2ITR-OV4-KLK1-injected hens than that in the control vector-injected birds. These data indicate that AAAV ITRs and/or Rep gene can promote KLK1 gene expression in an oviduct-specific manner without evidence for persistent expression.
To characterize the rhK1, egg white of the vector-injected laying hens submitted to Western blotting analysis using an antibody specific for human tissue kallikrein. The results showed that a specific protein band of 37kDa was detected, which corresponded to the mature enzyme. Further biochemical studies showed that the rhK1 had a similar thermo-stability, optimal pH and sensitivity to different ions to the natural enzymes from human and porcine tissues.
To establish a quantitative detection method for rhK1, by using affinity-purifed monoclonal antibody against hK1 as the coating antibody and the E.coli-expressed GST-KLK1 fusion protein as the known antigen, a sandwich ELISA was established. Under the optimized conditions, the ELISA reading values had intra- and inter-plate linear relationship with the antigen concentrations ranging from 16μg/ml to 125μg/ml. The egg whites of the vector–injected hens were submitted to the ELISA detection and the hK1 of 332, 476 and 248μg/ml was detected at days 3, 6 and 9 after vector injection, respectively.
引文
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4. Yates VJ, el-Mishad AM, McCormick KJ, et al. Isolation and characterization of an avian adenovirus-associated virus. Infect Immun, 1973, 7: 973-980.
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8. Bossis I, Chiorini JA. Cloning of an avian adeno-associated virus(AAAV) and generation of recombinant AAAV particles. J Virol, 2003, 77(12): 6799-6810.
9. Estevez C, Villegas P. Sequence analysis, viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus. Virus Research, 2004, 105: 195-208.
10.王建业,孙怀昌,朱国强.禽腺联病毒的分离及其基因组鉴定. 2005, 26(2): 1-4.
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19. Mayor HD, Torikai K, Melnick JL, et al. Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions. Science, 1969, 166: 1280-1282.
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23. Balagúe C, Kalla M, Zhang WW.Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.J Virol, 1997,71(4):3299-3306.
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2. Tsao J, Chapman MS, Agbandje M, et al. The three dimensional structure of canine parvovirus and its functional implications. Science, 1991, 251: 1456-1464.
3. Atchison RW, Casto BC, Hammon W. Adenovirus-associated defective virus particles. Science, 1965, 149: 754-756.
4. Yates VJ, el-Mishad AM, McCormick KJ, et al. Isolation and characterization of an avian adenovirus-associated virus. Infect Immun, 1973, 7: 973-980.
5. Bauer HJ, Schneider R, Gelderblom HR, et al. Biological and physicochemical characterization of the major (1.40) and minor (1.45) component of infectious avian adeno-associated virus. Arch Virol, 1991, 120: 123-133.
6. Hess M, Paul G, Kling S, et al. Molecular characterization of two strains of the avian adeno-associated virus (AAAV). Arch Virol, 1995, 140: 591-598.
7. Yates VJ, Dawson GJ, Pronovost AD. Serologic evidence of avian adeno-associated virus infection in an unselected human population and among poultry workers. Am J.Epidemiol, 1981, 113: 542-545.
8. Bossis I, Chiorini JA. Cloning of an avian adeno-associated virus(AAAV) and generation of recombinant AAAV particles. J Virol, 2003, 77(12): 6799-6810.
9. Estevez C, Villegas P. Sequence analysis, viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus. Virus Research, 2004, 105: 195-208.
10.王建业,孙怀昌,朱国强.禽腺联病毒的分离及其基因组鉴定. 2005, 26(2): 1-4.
11. Kotin RM. Site specific integration by adeno-associated virus. Proc Natl Acad Sci uSA, 1990, 87: 2211-2215.
12. Kotin RM, Linden RM, Berns KI. Characterization of a preferred site on humanchromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J, 1992, 11: 5071-5078.
13. Alexander IE, Russell DW, Miller AD. DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors. J Virol, 1994, 68: 8282-8287.
14.殷震,刘景华主编.动物病毒学. [M]科学出版社. 1997: 1145-1147.
15. Hermonat PL, Labow MA, Wright R, et al. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol, 1984, 51: 329-339.
16. McLaughlin SK, Collis P, Hermonat PL, et al. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol, 1988, 62: 1963-1973.
17. Carter BJ. Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective. Molecular Therapy, 2004, 10(6): 981-989.
18. Melnick JL, Mayour HD, Smith KO, et al. Associa tion of 20 millimicron particles with adenoviruses. J Bacteriol, 1965, 90: 271-274.
19. Mayor HD, Torikai K, Melnick JL, et al. Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions. Science, 1969, 166: 1280-1282.
20. Fu Y, Wang Y, Evans SM.Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus. Nat Biotechnol, 1998, 16(3):253-257.
21. Hsiao CD, Hsieh FJ, Tsai HJ. Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.Dev Dyn, 2001, 220(4):323-336.
22. Weitzman MD, Ky?sti? SR, Kotin RM, et al.Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci u S A, 1994, 91(13):5808-5812.
23. Balagúe C, Kalla M, Zhang WW.Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.J Virol, 1997,71(4):3299-3306.
24. Rinaudo D, Lamartina S, Roscilli G, et al. Conditional site-specific integration into human chromosome 19 by using a ligand-dependent chimeric adeno-associated virus/Rep protein. J Virol, 2000, 74(1):281-294.
25. Huttner NA, Girod A, Schnittger S.Analysis of site-specific transgene integration following cotransduction with recombinant adeno-associated virus and a rep encodingplasmid. J Gene Med, 2003, 5(2),120-129.
26.尹春光,杜立新.转基因鸡的研究策略.生物学通报,2004,39(3):13-15.
27. Dierich A, Gaub MP, LePennec JP, et al. Cell-specificity of the chicken ovalbumin and conalbumin promoters. EMBO J, 1987, 6(8): 2305-2312.
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