人睾丸特异表达基因PIAS-NY 原核表达 和多克隆抗体的制备

Prokaryotic expression of human testis-specific PIAS-NY and preparation of polyclonal antibody

摘要:

目的 构建PIAS-NY-pET30a 原核表达载体，并诱导其成功表达，经纯化后获得高浓度的可溶性融 合蛋白His-PIAS-NY，免疫小鼠获得鼠源性PIAS-NY 多克隆抗体。方法 以人精原cDNA 文库为模板，通过 聚合酶链反应（PCR）扩增PIAS-NY 开放阅读框，克隆到pET30a 表达载体中，酶切、PCR 鉴定构建重组质粒。 测序完全正确后将质粒PIAS-NY-pET30a 转化到大肠杆菌BL21（DE3）中进行大量扩增，异丙基-β-D- 硫 代半乳糖苷（IPTG）诱导表达。超声、离心、His-Ni 柱亲和层析纯化包涵体蛋白，依次经过不同浓度尿素进 行蛋白质复性，获得高浓度的可溶性His-PIAS-NY 融合蛋白。分6 次BalB/C 小鼠腹腔注射乳化纯化蛋白。 2 个月后小鼠眼球取血，收集血清。结果 成功构建原核表达质粒PIAS-NY-pET30a ；利用终浓度1 mmol/L IPTG 37℃诱导4 h，大肠杆菌BL21 大量表达融合蛋白His-PIAS-NY，并以包涵体形式存在于菌体中；尿素 变性后His-Ni 柱纯化融合蛋白，复性后获得高浓度的可溶性蛋白His-PIAS-NY ；6 次免疫后收获存活小鼠 抗血清；酶联免疫吸附测定（ELISA）抗血清滴度达到1 ∶ 100 000 ；免疫印迹和免疫共沉淀证实PIAS-NY 抗血清能特异性识别293T 细胞和GC2 细胞中PIAS-NY 蛋白。结论 成功获得PIAS-NY 多克隆抗体，而且 具有高反应性和高特异性，PIAS-NY 在GC2 细胞和293T 细胞中表达，并且能够与RUVBL2 蛋白发生相互作用。

Abstract:

Objective To construct a recombinant prokaryotic vector inducibly expressing human PIASNY, and prepare mouse polyclonal antibody against His-PIAS-NY. Methods PIAS-NY gene was amplified by PCR with human spermatogonial cDNA library as a templation. PCR products were inserted into PET-30a vector. The recombinant plasmid was successively verified by PCR, enzyme digestion and DNA sequencing analysis. The Escherichia coli BL21 (DE3) containing recombinant vector PIAS-NY-pET30a was induced to express fusion protein by IPTG. After denaturation, protein was purified with Imidazole elution by affinity chromatography followed by protein refolding. Mice were immunized by peritoneal injection of recombinant PIAS-NY protein for six times.Polyclonal antibody was obtained by harvesting mouse serum, bioactivity of which was examined by ELISA. Results The recombinant prokaryotic expression vector was successfully constructed. Expression of fusion protein was induced by IPTG induction at 37℃ for 4 hours in the form of inclusion bodies in Escherichia coli (BL21). Highly-purified soluble recombinant protein His-PIAS-NY was obtained after denaturation, purification and dilution renaturation. Serum levels of specific anti-PIAS-NY polyclonal antibody obtained from mouse serum was detected by ELISA at titer above 1: 100000. Western blot and co-immunoprecipitation assay confirmed its high specificity. Conclusions The polyclonal antibody against human PIAS-NY is obtained; it can recognize the protein within GC2 and 293T cells, which will be helpful for further study in spermatogenesis.