鼠抗重组人跨膜四蛋白33抗体的制备及其研究

Preparation and characteristic of mouse anti-human recombinant TSPAN33 antibody

目的制备鼠抗重组跨膜四蛋白33（TSPAN33）抗体并验证TSPAN33在桥本氏甲状腺炎外周血B细胞中的表达。方法通过PCR法从霍奇金淋巴瘤细胞的cDNA中扩增TSPAN33基因，并构建到pET32a表达载体中。使用IPTG诱导重组蛋白的表达，通过镍柱亲和层析纯化获得TSPAN33蛋白。以纯化的TSPAN33蛋白混合福氏完全佐剂免疫Balb／e小鼠制备抗体，通过合成TSPAN33胞外区肽段耦联环氧基磁珠，亲和富集抗TSPAN33胞外区特异性IgG，洗脱得抗体用酶联免疫吸附实验（ELISA）和Westernblot分别检测效价和特异性。同时使用制备的抗体初步对桥本氏甲状腺炎（HT）患者外周血TSPAN33＋B细胞比例进行检测。结果成功的表达、纯化了TSPAN33重组蛋白，抗TSPAN33蛋白免疫后血清通过固相肽段亲和富集纯化后抗体效价为1：64000，Westernblot和流式细胞术分析显示该纯化抗体不但能够与TSPAN33蛋白特异性的结合，同时还能够和天然的TSPAN33结合，流式细胞术结果显示HT患者外周血TSPAN33＋B细胞比例为（4．71±0．43）％，高于健康组（1．95±0．36）％（P〈0．01）。结论以重组TSPAN33为免疫原，成功地制备高效价、高特异性的兔抗TSPAN33抗体，为进一步进行TSPAN33的检测及其临床应用研究奠定了基础。

Objects To prepare the mouse anti recombinant human tetraspanin 33 （TSPAN33） antibody and to determine the ratio of TSPAN33 ＋ B cells in B cells in peripheral blood. Methods The gene coding of TSPAN33 was amplified by PCR from the cDNA of Hodgkin＇s lymphoma cells and cloned into prokaryotic ex- pression vector pET32a. The recombinant plasmid pET32a/TSPAN33 was transformed into E. eoli BL21 （ DE3 ） and expressed under IPTG induction. The recombinant TSPAN33 was purified through Ni2 ＋ -NT agarose gel col- umn and the purified TSPAN33 was used as imunogen to imunize the mouse. The immune serum polyelonal anti- body was purified by peptide affinity chromatography, peptide of extraeellular domain of TSPAN33 combined ep- oxy modified magnitie beads. The titer and specificity of purified antibody were analyzed by ELISA, WB and Flow cytometry （FCM） repeetively. Results The recombinant TSPAN33 was successfully expressed and puri- fied, and the anti-TSPAN33 polyclonal antibody was prepared successfully. The antibody for the peptide of extra- cellular domain of TSPAN33 was purified by affinity chromatography. The titer of the purified antibody wasl ： 64 000 determined by ELISA. Western blot and FCM analysis showed that the purified antibody reacted specific- ally to recombinant TSPAN33 and native TSPAN33. The ratio of TSPAN33 ＋ B cells in B cells from HT peripheral blood （4. 71 ± 0. 43 ） % was higher than healthy donors （ 1.95 ±0. 36） % by flow cytometry Method （FCM） （P 〈 0.05）. Conclusions The purified anti-TSPAN33 antibody with high titer and specificity has been successfully prepared. This antibody lays the foundation for further research in detection of TSPAN33 and its application.