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Construction of prokaryotic expression vector for human carboxypeptidase H and its application
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF DIABETES
- Issue:
- 2
- DOI:
- 10.3321/j.issn:1006-6187.2007.02.008
- Key Word:
- 糖尿病,自身免疫性;羧基肽酶H;重组蛋白质类
Abstract： 目的 构建人羧基肽酶H(CPH)的原核表达系统,以获得其重组蛋白,并初步应用于人CPH自身抗体(CPH Ab)的检测.方法 采用PCR方法,从质粒pcDNAⅡ扩增出CPH基因片段,将其克隆到原核表达载体pQE30后转化宿主菌E.coli M15,IPTG诱导CPH蛋白表达,用NiNTA亲和层析柱纯化,Western-blot法检测表达蛋白的抗原性.用重组CPH蛋白作为包被抗原,初步建立检测CPH Ab的ELISA方法.结果 获得了CPH原核表达载体,CPH蛋白得到了表达.纯化的蛋白可被CPH-Ab阳性的1型糖尿病患者血清识别.与放射配体法相比较,以重组CPH蛋白作为包被抗原初步建立的ELISA方法,其灵敏性和特异性均升高,分别为93.3%和97.5%.结论 重组CPH蛋白具有良好的抗原性,为糖尿病患者CPH Ab的检测奠定了基础.
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