摘要
目的通过遗传工程获得ＺＰ３α融合蛋白，以便用表达产物制备ＺＰ３α单克隆抗体。方法根据重新测序的猪ＺＰ３αｃＤＮＡ５＇端非编码碱基数，ｐＷＲ４５０表达质粒家族的ｐＷＲ４５０－２被选用来表达β－半乳糖苷酶（ＬａｃＺ）／ＺＰ３α融合蛋白。带５＇端非编码区和信号顺序的全长ＺＰ３αｃＤＮＡ片段，用ＥｃｏＲⅠ酶切自ｐＺ５８质粒，然后被重组插入ｐＷＲ４５０－２质粒中被截短ＬａｃＺ＇基因的３＇端多克隆区。此重组质粒转化大肠杆菌ＴＧ１后，ＺＰ３α融合蛋白的表达通过ＩＰＴＧ诱导。结果在１ｍｍｏｌ／ＬＩＰＴＧ存在下可观察到ＬａｃＺ／ＺＰ３α融合蛋白在大肠杆菌中的表达，表达量约占总细菌蛋白的５％。在ＳＤＳ－ＰＡＧＥ上，融合蛋白显示相对分子质量为１０．２×１０４。在蛋白印迹实验中，融合蛋白显示了与兔抗猪ＺＰＩｇＧ的特异免疫学反应。
Objective The zona pellucida (ZP) surrounding pig oocytes is the unique extracellular matrix composed of three sulfated glycoproteins (ZP1, ZP3α and ZP3β). It has been demonstrated that the ZP3α possesses sperm binding activity and cross reactive antigenicity with human ZP in vitro . Therefore, the ZP3α has been thought to be a potential immunogen for developing human contraceptive vaccine. Because it is very difficulty to isolate natural ZP3α, the main objective of the present study is to obtain ZP3α fusion protein by genetic engineering in order to make monoclonal antibodies against ZP3α by using the expression protein. Methods According to the 5' end noncoding base pair number of pig ZP3α cDNA which was re sequenced, the pWR450 2 vector of pWR450 expression plasmid family was chosen to express β galactosidase (LacZ)/ZP3α fusion protein. The 1700bp fragment of ZP3α cDNA with 5' end noncoding region and signal sequence was excised from plasmid pZ58 by EcoRI digestion and inserted into the 3' end polyclonging region of the truncated Lac Z' gene (1395bps) in pWR450 2 plasmid. Then, the recombinant plasmid was transformed into E.coli TG1 and the expression of the fusion protein was induced by IPTG. Results The re sequenced data showed that there was a base T missing between position 20 and 19 in the published 5' end noncoding region of ZP3α cDNA. The expression of LacZ’/ZP3α fusion protein in E.coli TG1 was observed at 1mmol/L IPTG, which the expressed product amount was about 5% of the total bacterial protein. On the SDS PAGE gel, the fusion protein display ecular weight of about 102kD matching with its deduced molecular mass. In addition, the fusion protein showed specific immunological reaction with anti pig ZP IgGs of rabbit in Western blotting test. This indicated that the expressed product was fusion protein with antigenic activity of pig ZP3α protein. Conclusion Collectively, the availability of pig ZP3α fusion protein will further help in making monoclonal antibody against ZP3α and evaluating its efficacy for fertility regulation.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1999年第2期137-141,共5页
Chinese Journal of Microbiology and Immunology
基金
世界卫生组织人类生殖研究
发展和培训特别规划署(WHO-HRP)及国家教育委员会留学回国人员科研费资助
