太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of antifreeze protein gene AFP4 in Pacific cod Gadus macrocephalus

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。

The open reading frame （ ORF） of type Ⅳ antifreeze protein was cloned from Pacific cod Gadus macro-cephalus using RT-PCR to investigate the function of type Ⅳ antifreeze protein. The result showed that the ORF of AFP4 had 375 bp in length, encoding a polypeptide of 125 amino acids. T h e recombinant pET-32a- AFP4 was constructed by linking the AFP4 ORF with the pET-32a vector, and transformed into Escherichia coli BL21 （DE3）. The expressing conditions of the recombinant plasmid were optimized under various concentrations of ther-mal hysteresis protein （ THP）,induction time and temperature with the optimal expressing induction conditions of 0.01 mmol/L IPTG,3 h and 37 ℃ .T h e recombinant protein was purified and used to immunize New Zealand white rabbit. The polyclonal antibody was obtained and tested using indirect ELISA, with the titer of 1 ： 1 600 000. The specific binding between recombinant protein and Rabbit anti-AFP4 polyclonal antibody indicated that the recombinant protein had high immunogenicity. The findings will provide the foundations for further under-standing of AFP4 functions in Pacific cod.