發(fā)布時間：2018-08-24 20:50

【摘要】：為獲得豬流行性腹瀉病毒(PEDV)受體豬氨基肽酶N(pAPN)抗原,提取巴馬香豬小腸絨毛上皮的總RNA,PCR擴增獲得pAPN基因的主要抗原區(qū)片段,并將其克隆至原核表達載體pET-32a中,將重組質(zhì)粒pET-32a-pAPN轉(zhuǎn)化大腸埃希菌BL21,通過不同濃度的IPTG、不同誘導(dǎo)時間進行誘導(dǎo)表達,以確定目的蛋白表達的最佳條件。經(jīng)SDS-PAGE和抗組氨酸標(biāo)簽的單抗進行Western blot檢測重組蛋白在大腸埃希菌中的表達,結(jié)果顯示,原核表達產(chǎn)物在誘導(dǎo)溫度37℃、IPTG濃度為0.8mmol/L,誘導(dǎo)4h可獲得最佳表達,產(chǎn)物分子質(zhì)量約為45ku,獲得的目的蛋白大小與預(yù)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，

本文編號：2202001