重组人巨细胞病毒嵌合肽的表达纯化及活性研究

发布时间：2018-01-15 12:12

本文关键词：重组人巨细胞病毒嵌合肽的表达纯化及活性研究　出处：《暨南大学》2005年硕士论文　论文类型：学位论文

【摘要】：人巨细胞病毒(human cytomegalovirus,HCMV)感染在人群中非常普遍,主要是呈隐性或亚临床感染,近年来发现该病毒不只在新生儿、免疫抑制及免疫缺陷患者中导致广泛的临床症状和死亡,而且可能与人类三大疾病--心血管疾病,恶性肿瘤及糖尿病有关,因而建立HCMV的快速检测方法具有重要意义。多种HCMV病毒蛋白可刺激机体产生相应的抗体,选用抗原性强、特异性好的蛋白抗原代替全病毒以检测HCMV感染,是提高HCMV感染诊断的敏感性和特异性的有效途径。国外的研究证实,HCMV的gp52蛋白和pp150蛋白具有较强的抗原性,在HCMV感染者的血清中其相应的抗体检出率较高,本实验室已成功构建串连有编码gp52 C末端和编码pp150 C末端两个基因片段的pPIC9K-rHCMVp表达质粒,并在毕赤酵母获得成功表达。但该质粒缺少纯化标记,不利于分离纯化,本研究将该质粒中人巨细胞病毒嵌合肽(rHCMVp)基因片段导入带纯化标志his6的pPICZαA中,重新构建、筛选了表达人巨细胞病毒嵌合肽(rHCMVp)的基因工程酵母。根据其基因序列,设计引物从pPIC9K-rHCMVp上扩增得到目的基因片段,并导入毕赤酵母诱导型表达载体pPICZαA中。通过电击将线性化的重组质粒转化到毕赤酵母GS115细胞中,筛选获得表达量较高的重组菌株,研究了该菌株生长的培养条件,包括不同诱导时间、甲醇浓度、pH值对人巨细胞病毒嵌合肽表达的影响。2L发酵罐进行了高密度发酵,经1%的甲醇、pH6.0的条件下诱导48h,最终菌体密度OD_(600)达到180,每升发酵液中含目的蛋白78.7mg,比摇瓶提高了4.8倍的产量。rHCMVp嵌合肽蛋白可通过高密度发酵大量获得。通过金属螯合亲和层析分离纯化发酵液上清,获得较高纯度的rHCMVp嵌合肽蛋白,进行了活性研究。实验结果证实该蛋白能被抗全病毒血清检测,且具有免疫原性;与市售进口的ELISA人巨细胞病毒检测试剂盒相比,两种方法的一致性较好,我们选择的gp52和pp150两个片段的嵌合肽蛋白作为包被抗原可用于HCMV感染的检测,为制备或组装高特异性和敏感性的HCMV基因工程抗原试剂盒提供依据。
[Abstract]:Human cytomegalovirus (HCMV) infection is very common in the population, mainly in the form of recessive or subclinical infection. In recent years, it has been found that the virus not only causes a wide range of clinical symptoms and deaths in newborns, immunosuppressive and immunodeficient patients, but also may be associated with three major human diseases-cardiovascular disease, malignant tumor and diabetes. Therefore, it is of great significance to establish a rapid detection method for HCMV. A variety of HCMV virus proteins can stimulate the body to produce corresponding antibodies, and select strong antigenicity. It is an effective way to improve the sensitivity and specificity of the diagnosis of HCMV infection by replacing the whole virus with specific protein antigen. The gp52 protein and pp150 protein of HCMV have strong antigenicity, and the detection rate of the corresponding antibodies in the serum of HCMV infected patients is higher. We have successfully constructed a series of pPIC9K-rHCMVp expression plasmids with two gene fragments encoding the C-terminal of gp52 and the C-terminal of pp150. The plasmid was successfully expressed in Pichia pastoris, but the plasmid was not suitable for purification. In this study, the gene fragment of human cytomegalovirus chimeric peptide rHCMVpwas transferred into pPICZ 伪 A with purified his6, and was reconstructed. The recombinant yeast expressing human cytomegalovirus chimeric peptide rHCMVpwas screened. According to its gene sequence, primers were designed to amplify the target gene fragment from pPIC9K-rHCMVp. The linearized recombinant plasmid was transformed into Pichia pastoris GS115 cells by electroporation. The effects of methanol concentration and pH on the expression of chimeric peptide of human cytomegalovirus (HCMV) were studied. The culture conditions of the strain were studied, including the effects of different induction time and pH value on the expression of human cytomegalovirus chimeric peptide. Under the condition of pH6.0 for 48h, the final cell density of ODS600) reached 180, and the target protein was 78.7 mg per liter of fermentation broth. Compared with shaking flask, the yield of rHCMVp chimeric peptide protein was increased by 4.8 times, which could be obtained by high-density fermentation. The supernatant of fermentation broth was separated and purified by metal chelating affinity chromatography. The high purity rHCMVp chimeric peptide protein was obtained and its activity was studied. The results showed that the protein could be detected by antiviral serum and had immunogenicity. Compared with the imported ELISA human cytomegalovirus detection kit, the two methods are consistent. We selected the chimeric peptide proteins of gp52 and pp150 as coated antigens for the detection of HCMV infection. To provide the basis for the preparation or assembly of highly specific and sensitive HCMV gene engineering antigen kit.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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