太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备
发布时间：2018-11-13 13:05
【摘要】：为进一步研究太平洋鳕Gadus macrocephalus抗冻蛋白AFP4的功能,通过RT-PCR方法克隆得到太平洋鳕4型抗冻蛋白基因(AFP4)的开放阅读框(open reading frame,ORF),构建原核表达载体并优化表达条件,利用纯化的重组蛋白制备多克隆抗体,采用间接ELISA法检测抗体的效价,用Western-blot法分析重组蛋白的免疫原性。结果表明:AFP4基因编码区长度为375 bp,编码125个氨基酸;将AFP4基因的ORF与载体p ET-32a连接,构建重组体p ET-32a-AFP4,并将其转入大肠杆菌BL21(DE3)感受态细胞中;经诱导、表达和条件优化,发现该重组体在37℃、0.01 mmol/L IPTG条件下诱导3 h获得最大的表达量;对该重组蛋白进行纯化,利用纯化的蛋白免疫新西兰大白兔制备多克隆抗体,采用间接ELISA法检测该抗体效价为1∶1 600 000;Western blot分析显示,重组蛋白可以与兔抗AFP4多克隆抗体发生特异性结合,表明该重组蛋白具有免疫原性。本研究结果可为深入研究太平洋鳕AFP4蛋白功能提供理论依据。
[Abstract]:In order to further study the function of Pacific cod Gadus macrocephalus antifreeze protein AFP4, the open reading frame (open reading frame,ORF of Pacific cod type 4 antifreeze protein gene (AFP4) was cloned by RT-PCR method, and the prokaryotic expression vector was constructed and the expression conditions were optimized. The polyclonal antibody was prepared from the purified recombinant protein, the titer of the antibody was detected by indirect ELISA method, and the immunogenicity of the recombinant protein was analyzed by Western-blot method. The results showed that the encoding region of AFP4 gene was 375 bp, encoding 125 amino acids, the ORF of AFP4 gene was ligated with the vector p ET-32a, and the recombinant pET-32a-AFP4, was constructed and transferred into E. coli BL21 (DE3) competent cells. After induction, expression and condition optimization, it was found that the recombinant obtained the maximum expression at 37 鈩，
本文编号：2329194
[Abstract]:In order to further study the function of Pacific cod Gadus macrocephalus antifreeze protein AFP4, the open reading frame (open reading frame,ORF of Pacific cod type 4 antifreeze protein gene (AFP4) was cloned by RT-PCR method, and the prokaryotic expression vector was constructed and the expression conditions were optimized. The polyclonal antibody was prepared from the purified recombinant protein, the titer of the antibody was detected by indirect ELISA method, and the immunogenicity of the recombinant protein was analyzed by Western-blot method. The results showed that the encoding region of AFP4 gene was 375 bp, encoding 125 amino acids, the ORF of AFP4 gene was ligated with the vector p ET-32a, and the recombinant pET-32a-AFP4, was constructed and transferred into E. coli BL21 (DE3) competent cells. After induction, expression and condition optimization, it was found that the recombinant obtained the maximum expression at 37 鈩，
本文编号：2329194
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