黄牛Ghrelin基因的克隆、表达及生物学活性检测

Cloning, prokaryotic expression of cattle Ghrelin gene and biological activity detection of the expressed protein

摘要:

本研究通过RT-PCR方法从秦川牛皱胃胃底腺mRNA扩增获得Ghrelin基因的cDNA序列, 克隆至pGM-T载体获得重组质粒pGh-T1, 并进行序列测定。将测序正确的cDNA序列定向克隆到pET32a(+), 构建表达载体pGh-32, 并转化BL21(DE3)大肠杆菌。IPTG诱导后SDS-PAGE分析表明秦川牛Ghrelin基因在大肠杆菌中获得高效表达, 可溶性分析表明Ghrelin融合蛋白以可溶性和包涵体2种形式表达。Western blotting初步证实了所获的融合蛋白是特异的。镍柱亲和层析法分离纯化融合蛋白并皮下注射家兔后获得了融合蛋白的抗体血清, ELISA检测血清的稀释效价在1:12 800。将获得的血清与黄牛下丘脑弓状核进行免疫组化试验, 进一步印证了重组蛋白表达产物是有活性的, 为进一步研究黄牛Ghrelin的功能及其对黄牛生长发育和脂肪沉积奠定了基础。

Abstract:

The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGEM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a(+) and transformed into host Escherichia coli strain BL2l (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western-blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.