摘要
运用RT-PCR方法,获得籽鹅脑垂体催乳素(Prolactin,PRL)基因编码区序列cDNA,克隆到pMD18-T载体上。DNA序列分析表明,PRL cDNA全长690 bp,编码230个氨基酸残基,与皖西白鹅的碱基同源性达99.57%,氨基酸同源性达99.56%。将所得序列与表达载体pET-32 a(+)构建表达质粒pET-32 a(+)-PRL。该质粒的DH5α转化菌经终浓度为1 mmoL.L-1的IPTG在37℃环境下诱导5 h可表达PRL基因融合蛋白,表达量约占菌体总蛋白的29.63%。用纯化的融合蛋白和凝血酶酶切后的单体蛋白主动免疫小白鼠,经Western blot检测,所制抗体清晰地检测到PRL基因表达蛋白的特异带,由此证明PRL基因的融合蛋白和凝血酶酶切后的单体蛋白均有较好的免疫原性。
The mature segment gene of prolactin（PRL） in Zi Goose was amplified from pituitary by RT-PCR and then cloned into the pMD18-T vector.Sequencing analysis showed that the cDNA has a length of 690 bp and encodes a protein composed of 230 amino acid residues,which differs from the published PRL cDNA sequence.There was a homology of 99.57% in base and 99.56% in amino acid residues with that of Wanxi White Goose,respectively.A prokaryotic expression vector,pET-32 a（＋）,was used to construct the recombinant plasmid pET-32 a（＋）-PRL to produce protein.Having been induced by IPTG,the host cell carrying the recombinant plasmid expressed the recombinant PRL.The optimal condition for expression was 1 mmoL·L-1 IPTG at 37 ℃.Based on this condition,the expression dose rose to the highest level by 5 hours of induction,accounting for 29.63% of the total bacterial protein.Purified fused proteins and digested PRL monomers by thrombin were injected into Kuming mouse to perform active immunity,the results showed that the fused proteins of PRL could be tested clearly by westernblot,and both of fused proteins and monomers have efficient immunogenicity.
出处
《黑龙江八一农垦大学学报》
2011年第6期31-35,共5页
journal of heilongjiang bayi agricultural university
基金
黑龙江省科技厅攻关项目(GB06B204-4)
