猪ZP4B细胞表位人源化定点突变

Production of humanized porcine zona pellucida (ZP)-4 B cell epitope by site-directed mutagenesis

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.