论文部分内容阅读
目的:构建人RGS22真核表达载体,表达人RGS22蛋白,制备其多克隆抗体,以探讨其在精子发生和肿瘤转移中的作用。方法:以cDNA文库为模板,用PCR法扩增人的RGS22基因。将其克隆到原核表达质粒PET22b中,构建重组质粒PET22b-RGS22。将该重组质粒依次进行PCR、酶切鉴定及测序后,转化BL21菌,以IPTG诱导表达。表达产物用质谱鉴定。纯化蛋白免疫小鼠,制备多克隆抗体,ELISA测定滴度。结果:用PCR法扩增出3 800 bp的RGS22基因编码框,测序证实为人RGS22基因,并在BL21菌中表达RGS22重组蛋白,用亲和层析N i柱进行纯化得到人RGS22蛋白。质谱分析其肽混合物的肽质量指纹谱,数据库检索证实为RGS22蛋白。用纯化的RGS22重组蛋白免疫小鼠2个月后,得到相应的多克隆血清抗体,经ELISA检测,滴度均能达到1/10万。结论:制备了RGS22的多克隆抗体,为进一步研究RGS22在精子发生中的作用创造了有利条件。
OBJECTIVE: To construct human RGS22 eukaryotic expression vector and express human RGS22 protein to prepare its polyclonal antibody to explore its role in spermatogenesis and tumor metastasis. Methods: The cDNA library was used as a template to amplify human RGS22 gene by PCR. It was cloned into prokaryotic expression plasmid PET22b to construct recombinant plasmid PET22b-RGS22. The recombinant plasmids were sequentially PCR, restriction enzyme digestion and sequencing, then transformed into BL21 strain and induced by IPTG. The expression product was identified by mass spectrometry. The purified protein was used to immunize mice, polyclonal antibodies were prepared and titers were determined by ELISA. Results: The coding region of RGS22 gene of 3 800 bp was amplified by PCR and confirmed to be human RGS22 gene. The recombinant protein of RGS22 was expressed in BL21 and purified by affinity chromatography on N i column to obtain human RGS22 protein. Mass spectral analysis of peptide mass fingerprinting of peptide mixtures, database search confirmed RGS22 protein. Two months after the mice were immunized with the purified RGS22 recombinant protein, the corresponding polyclonal serum antibodies were obtained, and the titer was estimated to be 1/10000 by ELISA. Conclusion: The polyclonal antibody of RGS22 was prepared, which provided favorable conditions for further study on the role of RGS22 in spermatogenesis.