Expression, Purification And Activity Of The Recombinant Peptide Of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a very commonly pathogen which usually causes asymptomatic or sub-clinic infections in the host. Recent studies have shown that HCMV not only lead to broad clinical symptoms and death in newborns, immunocompromised and immunodeficiency patients,but also can be associated with three major human diseases, cardiovascular disease, malignant tumour and diabetes. The rapid and accurate diagnosis of HCMV infections by the clinical laboratory plays an important role in the prevention and monitoring of treatment of these infections. Many proteins of HCMV can stimulate the organism to produce the corresponding antibody, so choose the antigens with strong immunoreactive and specific to replace the whole virus for detection of HCMV infection is the effective way of improving the sensitivity and specificity of HCMV infection. Some researchers have evidenced that the protein of HCMV gp52 and ppl50 are the strong immunoreactive antigens and their corresponding antibody have a relatively high detection rate in HCMV infection serum. The recombinant plasmid pPIC9K-rHCMVp containing the gene sequence of the C-terminus of gp52 and ppl50 has been successful constructed and expressed in Pichia pastoris in our lab's primary researches. But the plasmid lacks the purification marker which is unfavorable to the protein purification, in this research, the gene fragment coding carboxy terminus of gp52 and ppl50 was cloned into pPICZαA which with a 6Xhis tag and genetically engineered Pichia pastoris which expressed the recombinant HCMV peptide was screened again. The gene sequence of the recombinant HCMV peptide was amplified from plasmid pPIC9K-rHCMVp, and then transmited into expression vector pPICZ α A. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115. Then a high level expression strain in flasks was screened by SDS-PAGE and Western-Blotting.The transformant strain growth conditions were optimized in shake flasks including methanolconcentration, pH and inducing time. Then the conditions were applied to the production of rHCMVp in high-density fermentation. The highest expression of rHCMVp was obtained when the Pichia pastoris were induced with 1.0% methanol in pH6. 0 culture medium for 72h in shake flasks. After the genetically engineered yeast were induced in the 2-liter fermentor for 48h, the OD600 of cells was 124. The concentration of rHCMVp in the supernatant was 78. 7mg/L that was 4. 8 times more than that induced in the shake-flask for 72h. A great deal of rHCMVp were obtained by high-density fermentation and high-purity rHCMVp were gained by metal-chelating affinity chromatography from fermentation suspensions. The results in this paper indicated that rHCMVp could be detected with the anti-HCMV sersum and had immunogenicity in mouse. The high-sensitivity and high-specificity were observed when using purificated rHCMVp as the coating antigen comparing with commercial anti-HCMV IgM ELISA kit (Italy), the veracity of the rHCMVp in detection HCMV IgM was high. These suggest that recombinant gp52-ppl50 expressed in Pichia pastoris may be useful for development a sensitive and specific techniques for detection of HCMV infection.