【作者】 孟文丽；

【导师】 许云禄；

【作者基本信息】 福建医科大学 ， 药理学， 2008， 硕士

【摘要】 目的采用基因工程技术,构建江浙短尾蝮蛇纤溶酶原激活剂(plasminogen activator of Gloydius brevicaudus venom, GBV-PA )原核核表达载体(pET42a(+)-GBV-PA),并在大肠杆菌中的表达。为编码GBV-PA的基因克隆在大肠杆菌细胞中进行有效表达,批量生产GBV-PA;同时为研究GBV-PA的基因和蛋白质、结构和功能的关系及其作用机制创造条件。方法根据已知竹叶青蛇毒纤溶酶原激活剂(TSV-PA)基因的核苷酸序列设计上游引物,含EcoRⅠ酶切位点,和下游引物,含XhoⅠ酶切位点。以提取的江浙短尾蝮蛇毒腺总RNA为模板,RT-PCR方法合成扩增其中的纤溶酶原激活剂基因序列,将GBV-PA基因定向克隆到原核表达载体pET42a(+)中,经EcoRⅠ/XhoⅠ双酶切鉴定后,热激转化至宿主菌BL21(DE3),测序鉴定。IPTG诱导rGBV-PA融合蛋白表达,SDS-PAGE凝胶电泳鉴定表达产物;His亲和层析柱纯化融合蛋白rGBV-PA, Western blotting方法鉴定表达产物的抗原性。结果1.RT-PCR扩增得到的cDNA为800bp左右,与预期GBV-PA基因大小基本一致。2.构建的表达质粒pET42a(+)-GBV-PA,经双酶切和测序鉴定证实GBV-PA基因已正确插入质粒载体的多克隆位点。得知GBV-PA的cDNA大小为777bp,推导出的氨基酸数目为259个,MW=27921。GBV-PA与TSV-PA核苷酸序列同源性为90.13%,氨基酸序列同源性为81.92%。3.在大肠杆菌BL21中成功表达融合蛋白rGBV-PA,SDS-APGE凝胶电泳显示rGBV-PA主要以包涵体形式存在,分子量约61.7kDa。4.纯化得到融合蛋白rGBV-PA,具有蝮蛇毒的抗原性。结论成功构建了GBV-PA基因的原核表达载体,并在大肠杆菌中表达,经His亲和层析柱纯化后,获得rGBV-PA融合蛋白具有蝮蛇毒的抗原性。这些结果为江浙短尾蝮蛇纤溶酶原激活剂作为溶栓药物的进一步研发提供实验依据。更多还原

【Abstract】 ObjectiveThe subject using gene techniques constructed the prokaryotic expression vector of pET42a(+)-GBV-PA (plasminogen activator of Gloydius brevicaudus venom,GBV-PA),and expressed recombinate GBV-PA(rGBV-PA) fusion protein in Escherichia coli(E. coli). These create the possibility for expression of GBV-PA cloning efficiently in E. coli and mass production of GBV-PA. These also provide the basis for study on the relationship of constitution between function and the pharmacological mechanism of GBV-PA.MethodA couple of specific primers were designed on the basis of known nucleotide sequence of TSV-PA(Plasminogen Activator of Trimeresurus stejnegeri Venom) . The forward primer contained EcoRⅠrestriction site,and the reward prime contained XhoⅠrestriction site. GBV-PA gene fragment was amplified from total RNA of Gloydius brevicaudus venom gland with RT-PCR. The PCR products were cloned and inserted into prokaryotic expression plasmid pET42a(+).Recombinant plasmid (pET42a(+)-GBV-PA)were identified by restriction enzymes digestion,then transformed into E. coli strain BL21(DE3). pET42a(+)-GBV-PA was identified by sequencing. Expression of rGBV-PA fusion protein induced by IPTG,and the expressed product was analyzed by SDS- PAGE .The rGBV-PA fusion protein was purified with His-Bind resin protein purification procedure. The antigenicity of expressed product was identified by Western blotting.Results 1.The cDNA in 800 bp was obtained by RT-PCR amplification,which was the same as GBV-PA gene.2.The GBV-PA gene was synthesized sueeessuflly and inserted into the vector PET42a(+).The recombinant expression plasmid pET42a(+)-GBV-PA was constructed. The size of cDNA sequence of GBV-PA gene was 777 bp which encoding 259 amino acids. MW=27921. Homologies of nucleotide sequence and amino acid sequence of GBV-PA between TSV-PA were 90.13% and 81.92%, respectively.3. The rGBV-PA fusion protein was expressed in E. coli BL21(DE3). SDS - PAGE showed that the fusion protein was expressed as inclusion body formation in E. coli BL21(DE3). MW was 61.7kD.4. The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Gloydius brevicaudus venom.ConclusionsThe prokaryotic expression recombinant plasmid pET42a(+)-GBV-PA were successfully constructed. The GBV-PA gene was expressed in E. coli BL21(DE3). The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Agkistrodon halys pallas venom. This can provide experimental basis for research and development of GBV-PA as thrombolytic drug.更多还原