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目的通过PCR技术扩增,将YPEL4基因亚克隆到pGEX-4T-1原核表达载体,经酶切及测序鉴定后,转化E.coli BL21大肠杆菌,用异丙基-β-D-硫代半乳糖苷(isopropy-β-D-thiogalactoside,IPTG)诱导融合蛋白表达。融合蛋白在细菌裂解液的包涵体和上清中都有较高的表达,故取其上清液用谷胱甘肽琼脂糖珠(Glutathione Sepharo-seTM4B)亲和纯化目的蛋白。将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,进行WesternBlot检测抗体及其效价。结果获得了高效价的特异性兔抗YPEL4多克隆抗体,为YPEL4基因进一步的功能研究奠定了基础。
OBJECTIVE: To amplify YPEL4 gene into pGEX-4T-1 prokaryotic expression vector by PCR amplification. After digestion and sequencing, YPEL4 gene was transformed into E.coli BL21 E.coli and treated with isopropyl-β- Isopropy-β-D-thiogalactoside (IPTG) induces the fusion protein expression. The fusion protein was highly expressed in the inclusion body and supernatant of the bacterial lysate. Therefore, the supernatant was used to affinity-purify the target protein with Glutathione Sepharo-seTM4B. The purified fusion protein was immunized New Zealand white rabbits to prepare polyclonal antibodies, Western blotting detection of antibodies and titers. The results obtained high titer of rabbit anti-YPEL4 polyclonal antibody, YPEL4 gene for further functional study laid the foundation.