The Human Cytomegalovirus (hcmv) Pp71 Gene Cloning, Sequencing And Expression Study

(Biochemistry and Molecular Biology)Directed by professor Li Tian-XianHuman Cytomegalorvirus(HCMV) PP7 1 gene codes the tegument protein . This protein could enhance the infectivity of HCMV and accelerate HCMV infectious cycle. The protein, as a stronge antigen, can induce organism to produce antibody to eliminate virus. In order to study the PP7 1 gene expression in E. coli and the function of the purified protein in HCMV propasation, then to provide scientific basis for research of HCMV pathogenic, diagnosis and treatment, The researches presented is this thesis studied the cloning, sequencing and expression of HCMV PP71 in E.coli , and the function of the purified protein in HCMV propasation .The completely HCMV PP7 1 gene was first amplified by PCR(polymerase chain reaction) from the HCMV, which was stored in China Center For General Viruses Cultuer Collection, After digested with restriction enzyme BamH I and HindIII, the fragment then was insertrd into the high level expression vector pET28a plasmid. The recombinant plasmid PP71-Pet28a was transformed into E.coli BL21(DE3). Sequenced that PP7 1 gene was a conservative gene , with homology of-4-4,~99.69percent with others PP7 1 gene in GeneBank. After inducing with IPTG, the PP7 1 protein was expressed at high level in bacteria and the size of the protein is about 40 kD , which corresponds to the theoretic molecular weight. The expressed protein was purified with SephadexG-75High Q sepharose eficiently with the purity of 9lpercent. The optimal condition to expressing the PP7 I proteinis was that: The value of 0D600 was 0.5?0.9,lnduce emperature was 37 0C, Induce time was 4 hour and IPTG chroma was O.4?.5mMIml. Purified PP71 protein, HCMV, Purified PP7 1 protein mixed with HCMV was at the same time used to infect the Human-Embryo-Lung Cell respectively and analyse the Cell-Pathology-Effect (CPE). The result showed the CPE of the cells infected with HCMV and PP7 1 protein was stronger than the CPE was checked under microscope of the cells infected by HCMV alone, This result indicated that PP7 1 protein expressed in bacteria can enhance the infectivity of HCMV and accelerate the infectious cycle. These results indicatedHCMV PP71 gene can be express at high level in E.coh, the expressed PP7 1 protein was biologic function, which can enhance HCMV infectivity.