LY3磷酸酶基因的原核表达及其单克隆抗体的初步制备

发布时间：2018-01-10 04:04

本文关键词：LY3磷酸酶基因的原核表达及其单克隆抗体的初步制备　出处：《安徽医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:在扩增、克隆人的LY3基因的基础上,构建相应的重组原核表达载体,原核表达含LY3的融合蛋白,并以此为免疫原,应用杂交瘤单克隆抗体技术制备LY3单克隆抗体,为将来进一步研究该基因的功能奠定基础。方法:根据LY3蛋白编码基因的序列,设计引物,PCR扩增LY3基因,通过限制性内切酶酶切、连接等亚克隆技术,利用pET-28a(+)质粒构建含LY3基因的原核表达载体,酶切、测序等鉴定,转化大肠杆菌后加IPTG诱导融合蛋白表达,SDS-PAGE分离表达的LY3蛋白,经Ni2+-NTA凝胶亲和层析纯化目的蛋白,用Western Blot方法检测重组蛋白的表达。以SDS-PAGE分离得到的融合蛋白为抗原,免疫Balb/c小鼠,利用B淋巴细胞杂交瘤技术制备单克隆抗体,并通过间接ELISA方法鉴定。结果:成功构建了含LY3基因的重组原核表达质粒pET-28a(+)-LY3,转化大肠杆菌经IPTG诱导后,Western Blot检测到LY3融合蛋白的表达。以SDS-PAGE分离得到的融合蛋白为抗原免疫Balb/c小鼠,间接ELISA方法鉴定结果表明得到稳定的单克隆抗体。结论:成功构建出LY3原核表达载体并在原核细胞中表达成包涵体,以此作抗原免疫小鼠,成功制备出LY3单克隆抗体。为进一步研究LY3的生物学功能奠定基础。
[Abstract]:Objective: to construct the recombinant prokaryotic expression vector based on the amplification and cloning of human LY3 gene, and to express the fusion protein containing LY3 in prokaryotic expression. LY3 monoclonal antibody was prepared by hybridoma monoclonal antibody technique, which laid a foundation for further study of the function of the gene. Methods: according to the sequence of LY3 protein coding gene, primers were designed. The LY3 gene was amplified by PCR and subcloned by restriction endonuclease digestion and ligation. The prokaryotic expression vector containing LY3 gene was constructed by using pET-28a () plasmid. The expression vector was identified by restriction endonuclease digestion and sequencing. The fusion protein was isolated and expressed by SDS-PAGE and then purified by Ni2-NTA gel affinity chromatography. The expression of recombinant protein was detected by Western Blot. The fusion protein isolated by SDS-PAGE was used as antigen to immunize Balb/c mice. Monoclonal antibodies were prepared by B lymphocyte hybridoma technique. Results: the recombinant prokaryotic expression plasmid pET-28a (pET-28a- LY3) containing LY3 gene was successfully constructed and transformed into Escherichia coli induced by IPTG. The expression of LY3 fusion protein was detected by Western Blot. The fusion protein isolated from SDS-PAGE was used as antigen to immunize Balb/c mice. Conclusion: the prokaryotic expression vector of LY3 was successfully constructed and expressed as inclusion body in prokaryotic cells, which was used as antigen to immunize mice. LY3 monoclonal antibody was successfully prepared, which laid a foundation for further study on the biological function of LY3.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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