狂犬病病毒受体P75^(NTR)原核表达的抗原性分析及多克隆抗体制备

Antigenicity analysis of rabies virus neurotrophin receptor P75 expressed in prokaryote and preparation of its polyclonal antibody

【目的】分析狂犬病病毒(Rabies virus,RV)神经营养因子受体P75(P75NTR)原核表达的抗原性及制备出多克隆抗体,为进一步研究RV与P75NTR的相互作用机制奠定基础。【方法】采用RT-PCR从感染Flury株的NA细胞中克隆P75NTR基因,选取P75NTR基因的418~681 nt和831~1080 nt两个区域(共513 nt,编码171个氨基酸),与p ET-32a(+)重组构建原核表达载体,然后转化BL21(DE3)感受态细胞进行表达,再以纯化的P75NTR重组蛋白免疫昆明小鼠制备抗P75NTR抗体,并以Western blotting和间接免疫荧光试验(IFA)检测其抗原性。【结果】以原核表达载体p ET-P75-513转化BL21(DE3)感受态细胞,经0.2 mmol/L IPTG诱导6 h可获得较高表达量的P75NTR重组蛋白,且主要以可溶性蛋白形式进行表达,可用Ni-NTA树脂分离柱进行纯化。经Western blotting和IFA检测,发现制备获得的抗P75NTR抗体能与NA细胞表面自然的P75NTR及转染BHK-21细胞表达的P75NTR发生良好反应,且抗体效价高,可达1∶16000。【结论】原核表达的RV受体蛋白P75NTR抗原性强,免疫小鼠获得的多克隆抗体特异性高,反应性良好。

[Objective ]The antigenicity of rabies virus （RV） neurotrophin receptor P75 （P75NTR） expressed in prokaryote was analyzed, and its polyclonal antibody （Pcab） were prepared, in order to lay the foundation for further research on mechanism of interaction between RV and P75NTR. [Method]The P75NTR gene was cloned from NA cells of Flury-infected strain by RT-PCR. A total of 513 nucleotides（nt） containing two domains（418-681 nt and 831-1080 nt） ofP75NTR gene were linked with pET-32a（＋） vector to construct recombinant prokaryotic expression plasmid. And the recombinant plas- mid was transformed into competent cell E. coli BL21 （DE3）, so as to express recombinant protein P75NTR. Then the puri- fied recombinant protein P75NTR was used for immunizing Kunming mouse, in order to prepare RV anti-P75NTR antibody. Finally, the antigenicity of P75NTR was detected by Western blotting and indirect fluorescent assay（IFA）. [Result]The results showed that the prokaryotic expression vector PET-P75-513 was transformed into competent cell BL21 （DE3）. The high expression quantity of recombinant protein P75NTR was obtained by inducing recombinant strain with 0.2 mmol/L IPTG for 6 h. The recombinant protein P75NTR was expressed in the form of soluble protein and purified by using Ni-NTA affinity chromatography. The results of Western blotting and IFA showed that the prepared anti-P75NTR Pcab could well react with natural P75NTR on the surface of NA cell and P75NTR expressed by transfected BHK-21 cell, with high antibody titer of 1：16000. [Conclusion]The expressed RV receptor protein P75NTR in prokaryote has high antigenicity. And the obtained anti-P75NTR Pcab from immunized mice has high antibody specificity and reactivity.