| Human cytomegalovirus (HCMV) is a major pathogen for human beings , active infection in immunocompromised patients may lead to severe and life-threatening illness. Furthermore, it causes severe sequelae (such as teratism and abortion) in newborn after primary infection of their mothers during pregnancy. Because infections either are asymptomatic or are accompanied by symptoms that are not specific for HCMV, laboratory techniques represent a decisive diagnostic approach.Diagnosis of HCMV infection can be obtained by virus isolation, detection of viral antigens, nucleic acids and serological tests, and these techniques have both advantages and disadvantages. Serological testing is widely used in the HCMV infection diagnosis. HCMV-specific immunoglobulin M (IgM) is a sensitive and specific indicator of active or recent HCMV infection. Detection of HCMV-IgM is most commonly done by using preparation of the virus in an Enzyme-linked immunosorbent assay (ELISA). However, despite its advantages in easy and quick operation as well as lower requirements for other equipments, the sensitivity, specificity and standardization due to different viral preparations need to be solved. Recombinant technology to produce the cloning and expression of HCMV antigen has gradually attracted scholars around the world. There are fewer reports about these in our country.There are several antigens which can detect HCMV- IgM ,and gp52 and pp150 are the most immunoreactive antigens.In this work, gene fragments containing antigenic epitopes of the carboxy terminus of gp52 gene and pp150 gene were cloned and expressed in E. Coli as fusion proteins with glutathione S-transferase (GST) and the antigenicity effusion proteins were identified. The study includes two parts: 1.The gp52 gene expression in E. Coli and the antigenicity study of recombinant protein; 2. The co-expression of pp150 gene and gp52 gene in E. Coli , the purification and theantigenicity study of the fusion protein.The gp52 gene fragment encoding epitope with combined IgM antibodies was amplified by PCR. The amplified PCR product was cloned into T-vector. After DNA sequenceing, the gp52 gene fragment was inserted into vector pGEX-5x-3 with GST fusion protein (the recombinant plasmid pGEX-gp52) and expressed in E. Coli. The results showed that the fusion gene was expressed effectively in pGEX-5x-3 vector/host system, and the fusion protein existed in soluble suspensions and inclusion bodies with molecular size of 41Kda proximately, which was corresponding to the estimated. Results of Western-blotting and indirect ELISA showed that the recombinant gp52 protein purified from Glutathione Sepharose 4B had good specific antigenic characterization, which could detect HCMV specific IgM in patient's sera. There was no impact on the activity of fusion antigen with the existence of GST.In the second part of study, pp150 strong antigenic gene fragment was amplified by PCR using primers, containing recognition sequences of the restriction endonucleases, to facilitate the following cloning steps. The amplified DNA fragment inserted into pGEX- gp52 expression vector directly after EcoR I and BamH I digestion, and then transformed into E. Coli. Through the sequencing of pGEX-pp150- gp52, it concluded that sequence of pp150-gp52 is the same as predicted. Engineering E.coli effectively expressing pp150 and gp52 recombinant protein was established. Indirect ELISA was established using simply purificated recombinant protein as the antigen coating polystyrene microdilution plates. ELISA plates were coated with 25ug/ml of GST-pp150-gp52 and 25ug/ml GST-gp52, respectively. Recombinant HCMV antigens were evaluated with 45 human serum samples, and compared commercial with the anti-CMV IgM ELISA kit (German Human). The result indicated that with GST- pp150- gp52 and GST-gp52 had distinctive coherence with the result of ELISA kit, the sensitivity of fusion antigens GST-pp150-gp52 and GST-gp52 was 83.9% and 74.1% respectively, the specificity was 85.7% and 78.6% respectively.The above study suggests... |
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