巴马香猪氨基肽酶N截短基因的克隆与原核表达
发布时间：2018-08-24 20:50
【摘要】：为获得猪流行性腹泻病毒(PEDV)受体猪氨基肽酶N(pAPN)抗原,提取巴马香猪小肠绒毛上皮的总RNA,PCR扩增获得pAPN基因的主要抗原区片段,并将其克隆至原核表达载体pET-32a中,将重组质粒pET-32a-pAPN转化大肠埃希菌BL21,通过不同浓度的IPTG、不同诱导时间进行诱导表达,以确定目的蛋白表达的最佳条件。经SDS-PAGE和抗组氨酸标签的单抗进行Western blot检测重组蛋白在大肠埃希菌中的表达,结果显示,原核表达产物在诱导温度37℃、IPTG浓度为0.8mmol/L,诱导4h可获得最佳表达,产物分子质量约为45ku,获得的目的蛋白大小与预期一致。
[Abstract]:In order to obtain the porcine aminopeptidase N (pAPN) antigen of porcine epidemic diarrhea virus (PEDV) receptor, the main antigen region of pAPN gene was amplified by total RNA,PCR amplification from the villus epithelium of Bama pig small intestine, and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmid pET-32a-pAPN was transformed into Escherichia coli BL21, and induced by different concentrations of IPTG, for different time to determine the optimal conditions for the expression of the target protein. The expression of recombinant protein in Escherichia coli was detected by Western blot with SDS-PAGE and anti-histidine labeled McAb. The results showed that the best expression could be obtained at 37 鈩，
本文编号：2202001
[Abstract]:In order to obtain the porcine aminopeptidase N (pAPN) antigen of porcine epidemic diarrhea virus (PEDV) receptor, the main antigen region of pAPN gene was amplified by total RNA,PCR amplification from the villus epithelium of Bama pig small intestine, and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmid pET-32a-pAPN was transformed into Escherichia coli BL21, and induced by different concentrations of IPTG, for different time to determine the optimal conditions for the expression of the target protein. The expression of recombinant protein in Escherichia coli was detected by Western blot with SDS-PAGE and anti-histidine labeled McAb. The results showed that the best expression could be obtained at 37 鈩，
本文编号：2202001
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