【摘要】：為進一步研究太平洋鱈Gadus macrocephalus抗凍蛋白AFP4的功能,通過RT-PCR方法克隆得到太平洋鱈4型抗凍蛋白基因(AFP4)的開放閱讀框(open reading frame,ORF),構(gòu)建原核表達載體并優(yōu)化表達條件,利用純化的重組蛋白制備多克隆抗體,采用間接ELISA法檢測抗體的效價,用Western-blot法分析重組蛋白的免疫原性。結(jié)果表明:AFP4基因編碼區(qū)長度為375 bp,編碼125個氨基酸;將AFP4基因的ORF與載體p ET-32a連接,構(gòu)建重組體p ET-32a-AFP4,并將其轉(zhuǎn)入大腸桿菌BL21(DE3)感受態(tài)細胞中;經(jīng)誘導(dǎo)、表達和條件優(yōu)化,發(fā)現(xiàn)該重組體在37℃、0.01 mmol/L IPTG條件下誘導(dǎo)3 h獲得最大的表達量;對該重組蛋白進行純化,利用純化的蛋白免疫新西蘭大白兔制備多克隆抗體,采用間接ELISA法檢測該抗體效價為1∶1 600 000;Western blot分析顯示,重組蛋白可以與兔抗AFP4多克隆抗體發(fā)生特異性結(jié)合,表明該重組蛋白具有免疫原性。本研究結(jié)果可為深入研究太平洋鱈AFP4蛋白功能提供理論依據(jù)。
[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，

本文編號：2329194