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猪卵透明带（PZP）是包绕在卵母细胞外的由ZPI、ZP3a和ZP35组成的一层丝状体酸性糖蛋白。其中的猪ZP39蛋白（分子量55kD，完全去糖基后为32kD）jfl当于小鼠精子受体ZP3和人ZP3蛋白，PZP35在受精过程中起何作用？特别是其抗体与猪精子受体PZP3a抗体一样也能在体外试验中阻断人精卵受精，所以长期以来PZP印也一直是生殖生物学和免疫避孕科研人员关注的研究对象。过去分离该蛋白组分是通过繁琐的生化提纯技术，随着分子生物学技术的广泛应用，以相对简便价廉的遗传工程方法大量制备无卵巢因子混杂的ZP目的蛋白已是势在必行。本文首次报道了PZP35基因在大肠杆菌中的表达，为以后制备相应的单克隆抗体，鉴别抗原决定簇进而研究ZP耿避孕疫苗打下了基础。一、目的基因的DNA测序为选择表达载体构建符合pZP30CDNA阅读框的融合基因，先用T。引物对pZ57质粒中PZP30基因的5’端重新作了DNA测序分析，结果发现已报道的该基因5’端非编码区漏读了二个碱基（T、G）。二、表达载体PWR450-2／ZP那的构建质粒PWR450-2带有裁短的LacZ’基因（编码6一半乳糖背酶N端461个氨基酸残基），外源基因插入LacZ’基因下游多聚克隆部位形成融合基因，以异丙基一p-Dwt代半乳糖着（IPTG）诱导上游的强启动子比c即产生高水平表达。将酶切
Porcine zona pellucida (PZP) is a layer of filamentous acid glycoprotein consisting of ZPI, ZP3a and ZP35 that surrounds the oocyte. Which porcine ZP39 protein (molecular weight 55kD, completely de-glycosylated after 32kD) jfl mouse sperm receptor ZP3 and human ZP3 protein, PZP35 in fertilization process play a role? In particular, their antibodies, like porcine sperm receptor PZP3a, can also block the fertilization of human sperm eggs in vitro. Therefore, PZP India has long been a research object of researchers in reproductive biology and immune contraception. In the past, the protein components were isolated through cumbersome biochemical purification technology. With the wide application of molecular biology techniques, it is imperative to prepare a large number of ZP protein without ovarian factor by a relatively simple and inexpensive genetic engineering method. This article first reported the expression of PZP35 gene in Escherichia coli, which laid the foundation for the future preparation of the corresponding monoclonal antibody, identification of antigenic determinants and further study of ZP Geng contraception vaccine. First, the DNA sequence of the target gene for the selection of expression vector construction consistent with the pZP30CDNA reading frame fusion gene, the first use of T Primer pairs of pZ57 plasmid PZP30 gene 5 ’end re-made DNA sequencing analysis and found that the gene has been reported 5’ non-coding region missed reading two bases (T, G). Construction of expression vector PWR450-2 / ZP Plasmid PWR450-2 carries a cloned LacZ ’gene (encoding 461 amino acid residues at the N-terminus of 6-galactose-6-lactase) and a foreign gene inserted downstream of the LacZ’ gene Cloning site to form a fusion gene, with isopropyl-p-Dwt generation of galactose with (IPTG) induced strong upstream of the promoter than c to produce high-level expression. Will be digested