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猪ZP4B细胞表位人源化定点突变 被引量:2

Production of humanized porcine zona pellucida (ZP)-4 B cell epitope by site-directed mutagenesis
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摘要 目的 :对编码猪ZP4的cDNA序列进行人源化定点突变 ,使原来编码LDPENLTL八肽表位的序列突变成编码相应人卵透明带上LDPEKLTL序列 ,同时保持其他序列不变。方法 :采用PCR重叠延伸法 ,连续进行 3次PCR ,对PZP4B细胞LD PENLTL八肽表位进行定点突变。第 3次PCR以前两次PCR的产物为模板 ,最终得到所需的突变体hpZP4和dpZP4。结果 :突变后的cDNA序列通过重组DNA技术 ,在突变体片段 5′及 3′末端分别引入SalI和NcoI内切酶位点 ,使其克隆入pBV2 2 1表达质粒中 ,制成hpZP4 pBV2 2 1,pZP4 pBV2 2 1和dpZP4 pBV2 2 1重组质粒。经测序 ,证明均为目的序列。结论 :通过定点突变产生人源化猪ZP4B细胞表位是可行的。研究将为人类克隆并制备新的人卵透明带避孕疫苗奠定基础。 Objective:To change the sequence of 8-mer epitope LDPENLTL of porcine zona pellucida (pZP)-4 into the corresponding fragments of LDPEKLTL in human ZP by site-directed mutagenesis of cDNA sequence.Methods:By double polymerase chain reaction (PCR), three PCR reactions were done. With former two PCR products as template, the mutated hpZP4 and dpZP4 were obtainted.Results:With recombinant plasmids coding native pZP4 as template, fragment hpZP4 containing the humanized epitope and dpZP4 fragment in which the epitope was deleted by an artificial frame shift mutation were obtained. The mutated cDNA sequences were cloned downstream of promoter P RRL in plasmid BV221(pBV221) through NcoI and SalI restriction sites for expression. Sequence analysis showed they are exactly the interest.Conclusion:Through humanized point mutation on porcine zona pellucida(pZP4), it's possible to find a novel way towards the development of a new B-cell epitope of ZP contraceptive vaccines which is of strong immunogenicity as well as potent antifertility effect.
出处 《中国免疫学杂志》 CAS CSCD 北大核心 2004年第3期184-186,共3页 Chinese Journal of Immunology
基金 复旦大学 985工程 (985B3 6) 上海市计生委科研基金 计划生育药具国家重点实验室课题 (B2 0 2 1)资助
关键词 卵透明带 B细胞表位 人源化定点突变 免疫避孕疫苗 Porcine zona pellucida B cell epitope Humanized point mutation Immunocontraceptive vaccines
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参考文献10

  • 1Minoru Shigeta,Akiko Hasegawa,Yukari Hamada et al.Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1)[].Journal of Reproductive Immunology.2000
  • 2Hasegawa A,Koyama K,Inoue M et al.Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters[].Journal of Reproductive Immunology.1992
  • 3Sun W,Lou Y H,Dean J et al.A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida[].Biology of Reproduction.1999
  • 4Govind C K,Hasegawa A,Koyama K et al.Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding[].Biology of Reproduction.2000
  • 5Barlow D J,Edwards M S,Thornton J M.Continuous and discontinuous protein antigenic determinants[].Nature.1986
  • 6Paterson M,Wilson M R,Morris K D et al.Evaluation of the contraceptive potential of recombinant human ZP3 and human ZP3 peptides in a primate model: their safety and efficacy[].American Journal of Reproductive Immunology.1998
  • 7Hasegawa A,Yamasaki N,Inoue M et al.Analysis of an epitope sequence recognized by a monoclonal antibody MAb-5H4 against a porcine zona pellucida glycoprotein(pZP4) that blocks fertilization[].Journal of Reproduction and Fertility.1995
  • 8Paterson M,Jennings Z A,Wilson M R et al.The contraceptive potential of ZP3 and ZP3 peptides in a primate model[].Journal of Reproductive Immunology.2002
  • 9Lou Y H,Bagavant H,Ang J et al.Influence of autoimmune ovarian disease pathogenesis on ZP3 contraceptive vaccine design[].Journal of Reproduction and Fertility.1996
  • 10Bradley M P,Eade J,Penhale J et al.Vaccines for fertility regulation of wild and domestic species[].Journal of Biotechnology.1999

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