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目的克隆鹦鹉热衣原体巨噬细胞感染增强蛋白(Microphage infectivity potentiator,Mip)基因,构建原核重组质粒,诱导表达重组蛋白,并免疫BALB/c小鼠,制备多克隆抗体,为进一步研究其功能奠定了基础。方法提取鹦鹉热衣原体6BC基因组DNA,PCR扩增Mip基因,克隆至p ET-30a(+),构建原核表达载体p ET-30a(+)-Cps Mip。PCR、酶切及测序鉴定后,IPTG诱导p ET-30a(+)-Cps Mip在E.coli BL21中表达目的蛋白。融合蛋白纯化后免疫BALB/c小鼠,ELISA检测小鼠血清中抗Mip抗体的效价。结果 PCR扩增得到大小约为768 bp左右的Mip目的片段;经PCR、酶切及测序鉴定,证明构建的原核重组质粒中插入的片段为Cps Mip基因,测序结果经BLAST分析,与Genbank上登录序列完全一致。经IPTG诱导,重组工程菌表达一相对分子量(Mr)约为34 k D的目的蛋白;ELISA检测免疫小鼠血清效价为1:128 000。结论成功构建了鹦鹉热衣原体Mip基因原核表达载体,并表达了相应可溶性蛋白,制备了高效价的鼠抗Mip多克隆抗体。
Objective To clone the gene of Microphage infectivity potentiator (Mip), construct the prokaryotic recombinant plasmid, induce the expression of recombinant protein, and immunize BALB / c mice to prepare polyclonal antibody, which laid the foundation for the further study of its function basis. Methods Genomic DNA of 6CC of Chlamydia psittaci was extracted, Mip gene was amplified by PCR and cloned into p ET-30a (+). The prokaryotic expression vector p ET-30a (+) - Cps Mip was constructed. After IPTG induction, p ET-30a (+) - Cps Mip was expressed in E. coli BL21 by PCR, restriction enzyme digestion and sequencing. The fusion protein was purified and immunized BALB / c mice. The titer of anti-Mip antibody in the serum of mice was detected by ELISA. Results The Mip fragment was about 768 bp in size. The PCR product was digested with restriction endonucleases and identified by sequencing. The inserted fragment was proved to be Cps Mip gene by BLAST analysis. The sequence is exactly the same. After induced by IPTG, the recombinant engineering bacteria expressed a protein of relative molecular weight (Mr) of about 34 kD. The serum titer of the immunized mice was 1: 128 000 by ELISA. Conclusion The prokaryotic expression vector of Mip gene of Chlamydia psittaci was successfully constructed, and the corresponding soluble protein was expressed. The polyclonal antibody against mouse polyclonal anti-Mip was prepared.