摘要
将鸡催乳素 (PRL)和抑制素 - α亚基 (INB- α)基因编码序列重组为融合基因 ,制备了同时包含这 2种激素基因的融合蛋白。通过 PCR和分子克隆的方法首先将全部粤黄鸡 PRL成熟肽 c DNA克隆到载体 p RSET A的 Bgl 和 Eco R 克隆位点之间 ,获得重组质粒 p PRL- RSET。鸡 INB- α片段经扩增后分别被克隆到质粒 p RSET A和 p PRL- RSET的 Nhe 和 Xho 克隆位点之间 ,获得重组质粒 p INB- RSET和 p INB- PRL。以上重组质粒构建的正确性分别由各特定引物组合扩增的 PCR产物长度、特定限制性内切酶消化各重组质粒所得产物长度以及对各质粒的测序结果得到验证。重组质粒 p PRL- RSET和 p INB- PRL 转化 E.coli BL2 1(DE3)株 ,IPTG诱导后所表达的产物经 SDS- PAGE显示 ,其分别与所预期的重组蛋白分子大小相符。质粒 p PRL - RSET和 p INB- PRL的表达产物和用 Ni- NTA凝胶纯化的 2重组蛋白产物都可与抗鸡 PRL 抗体产生特异的免疫印迹 ,并且表达菌裂解液和相应纯化蛋白的免疫印迹处于同一位置。结果说明 ,试验已成功完成了鸡 PRL、INB-α及 2者融合蛋白的构建。
In order to inhibit the broodiness and increase the egg laying performance in broody prone avians,the approach of producing recombinant proteins was chosen based on chicken prolactin and inhibin α subunit fragment.A cDNA sequence coding mature chicken prolactin molecule was amplified by PCR and cloned into cloning sites of BglⅡand EcoRⅠin plasmid pRSET A, which generated a recombinant expressing plasmid pPRL RSET.A fragment of chicken inhibin α subunit cDNA was also amplified and cloned into the sites of NheⅠand XhoⅠ in plasmids pRSET A and pPRL RSET,respectively to generate 2 recombinant expressing plasmids pINB RSET and pINB PRL.The accuracies of molecular clones in the recombinant plasmids were verified by PCR product lengths from specific primer combinations,by product length from specific restriction enzyme digestions and sequencing results of the plasmids.The 3 plasmids were each expressed in E. coli BL21(DE3) after induction by IPTG,and produced a proteinacous product showing the expected molecular size in SDS PAGE.The expressed products of pPRL RSET and pINB PRL were purified by Ni NTA Aagrose column, and expressed the same migration as their corresponding products in expressed bacterial lysates both in SDS PAGE and in Western blot shown by anti chicken prolactin antibody.These results demonstrated the successful construction of recombinant protein based on chicken prolactin and inhibin α subunit fragment.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2002年第4期365-367,共3页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目 (3 970 0 10 2 )
广东省自然科学基金资助项目 (970 0 13 [粤科基 (1997) 2 3 ] )
