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目的:克隆并构建PET-28a-GRA1重组表达载体,转化入大肠杆菌E.coli BL21,诱导表达并鉴定,为进一步研究GRA1的生物学特性和免疫保护作用奠定实验基础。方法:根据GenBank中编码GRA1的已知基因序列设计并合成一对引物,应用PCR技术扩增GRA1基因,插入原核表达载体PET-28a中,构建重组表达质粒PET-28a-GRA1,转化E.coli BL21,IPTG诱导后,进行SDS-PAGE和Western blotting鉴定。结果:克隆的GRA1基因与GenBank收录的基因序列同源性为100%。对重组质粒进行酶切和PCR鉴定,获得573bp大小的目的基因片段,与预期结果相符,成功地构建了重组质粒PET-28a-GRA1。SDS-PAGE电泳分析表明重组蛋白条带的相对分子质量约为24 000,Western blotting显示重组蛋白能被鼠抗弓形虫血清识别。结论:成功获取GRA1基因,构建了PET-28a-GRA1重组质粒并获得了高效表达。
OBJECTIVE: To clone and construct PET-28a-GRA1 recombinant expression vector and transform into E.coli BL21 for expression and identification, which will lay the foundation for further study on the biological characteristics and immunoprotection of GRA1. Methods: A pair of primers was designed and synthesized according to the known gene sequence encoding GRA1 in GenBank. The gene of GRA1 was amplified by PCR and inserted into the prokaryotic expression vector PET-28a. The recombinant plasmid was named as PET-28a-GRA1 and transformed into E.coli After induced by BL21 and IPTG, SDS-PAGE and Western blotting were used for identification. Results: The homology of the cloned GRA1 gene with that of GenBank was 100%. The recombinant plasmids were digested with restriction endonucleases and identified by PCR, and the target gene fragment of 573bp was obtained. Consistent with the expected results, the recombinant plasmid PET-28a-GRA1 was successfully constructed. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 24 000. Western blotting showed that the recombinant protein could be recognized by anti-Toxoplasma gondii serum. Conclusion: The GRA1 gene was successfully obtained and the recombinant plasmid PET-28a-GRA1 was constructed and highly expressed.