Parti. Human Cytomegalo Virus Expression And Purification Of Recombinant Proteins And Immunological Properties Partii. The Rubella Virus Igg Antibody Detection Method Research

Background:Human cytomegalovirus (HCMV; family Herpesviridae, subfamily Betaherpesivirinae, species Human herpesvirus5) is composed of the double-stranded liner DNA. HCMV is a successful, wide-spread pathogen that infects the majority of the world’s population. Although CMV is found throughout all geographic locations and in all socioeconomic groups, the infection rates are different. In Asia, approximately90%of the adult population is infected with CMV, and it’s usually produces asymptomatic infections in immunocompetent hosts. Serious disease can occur in immunocomopromised individuals (such the organ transplanted or HIV infected patients) and in congenitally infected newborns. Congenital HCMV infection can cause the damage of the fetal tiusses and organs when he/she was born (hepatosplenomegaly, petechiaeand, anemia et al), also can evoke a late complications (such as sensorineural hearing loss, mental retardation and visual impairment). In conclusion, Congenital HCMV infection is an important factor to cause the fetal defects at birth. Currently, there is no appropriate measure to treatment or prevent the infection of CMV. Accordingly, an effective antigen with high sensitivity and specificity, which can use for ELISA method, will play an important role in preventing the fetal defect and the HCMV spread.Objective:To obtain a soluble recombinant CMV antigen to detect the CMV antibody IgG in serum. Then, explore the immunological activity of this protein and forecast the possibility in the application for immunology detection. And, the results provide a theoretical basis for the next application.Methods:1. Synthesised CG3’s cDNA sequence, including three antigen epitopes of HCMV, pp150protein C-terminal (aa595～614and aa1006～1048) and p52protein C-terminus (aa202～433) and constructed CG3prokaryotic expression plasmid;2. Transformed the restructuring prokaryotic expression plasmid into E. coli BL21-(DE3), expressed CG3protein at the condition of16℃by IPTG in E. coli., BL- 21(DE3) strain, and purified the CG3protein by immobilized metal ion affinity chromatography (IMAC).3. Charaterized the immunologic properties of the recombinant antigen CG3protein by ELISA and Western-blotting.Results:1. Successfully constructed CG3prokaryotic expression vector and transformed the plasmid into E. coli BL21-(DE3);2. Determined the CG3protein expression conditions and solubility (partially soluble) by SDS-PAGE and Western-blotting.(The expression conditions included, the culture media:2YT, pH:7.0; the bacteria OD when added IPTG:0.5～1; IPTG:0.5mM; temperature:16℃);3. Determined the specificity of CG3protein by Western-blotting using positive and negative serum as the first antibody;4. Compared CG3protein with FDA-approved the SORIN anti-CMV IgG antibodies kit, the results showed that the CG3protein and Sorin Kit was moderate correlation, with sensitivity68.75%, specificity83.05%, false positive rate31.25%, false negative rate16.95%, coincidence rate86.82%, Kappa values0.52.Conclusions:1. The epitopes of recombinant antigen CG3can be identified by natural anti-cytomegalovirus IgG antibodies, and it also can be recognized by human anti-cytomegalovirus IgG antibodies;2. The recombinant antigen CG3, including HCMV pp150(UL32gene) and the pp52(UL44gene), has strong antigenicity.3. The recombinant antigen CG3protein with low sensitivity can effectively avoid reducing the specificity.4. The recombinant antigen CG3protein has a strong specificity to anti-HCMV-specific antibodies IgG. 5. The CG3protein with high-purity can be obtained by purification with a HisTrap column simply and conveniently.6. Recombinant protein CG3has application prospects.