Recombinant Expression Of Human Cytomegalovirus UL146 Gene Encoding Protein VCXCL-1 And Its Polyclonal Antibody Preparation And Identification

The life-long infection of human cytomegalovirus(HCMV)belongs to theβ-herpes virus family,and it is one of the most common public health threats.HCMV has a variety of immune evasion mechanisms to evade the host immune response to spread in the body.HCMV UL146,a HCMV open reading frame(ORF),encodes theα-chemokine vCXCL-1 which has been shown to facilitate viral latency and dissemination.However,a detailed molecular mechanism is still largely unclear due to the lack of reagents,such as vCXCL-1 specific antibodies.To further investigate the mechanism of vCXCL-1 involvement in evading the host immune system,the specific polyclonal antibodies were generated.This study provides the field an effective tool to unveil the role of UL146 in HCMV latency.In this study,polyclonal antibodies were prepared by immunizing New Zealand white rabbits with prokaryotic expression of the human cytomegalovirus UL146 gene encoding protein vCXCL-1 as follows:(1)The construction of recombinant plasmid expression vectors:HCMV BAC Towne library was used as template for UL146 gene PCR amplification,the PCR product was cloned into pET-32a(+).PCR products and vectors were double digested with Sal I and BamHI,ligated by T4 ligase,transformed into DH5 a E.coli,and plated on ampicillin plates.Positive clones were screened by double digestion and DNA sequencing.(2)Prokaryotic expression of recombinant protein: Recombinant plasmid pET32a(+)-UL146 was transformed into E.coli BL21(DE3),and the inducer was added to induce the expression of the recombinant protein in the prokaryotic cells,the expression conditions of recombinant protein including temperature,inducer concentration and time were optimized,and the expression pattern of the recombinant protein in prokaryotic which identificated by Western blot.(3)Purification of recombinant protein: a large number of recombinant proteins were induced to express under optimized conditions.The bacteria were breaked by sonication and dissolved with 8 M urea.Then the solubilized protein was separated and purified by affinity chromatography through Ni–NTA Resin,purified protein was renatured and desalted by dialysis.And the purity of the purified proteinwas analyzed by SDS-PAGE gel electrophoresis combined with a grayscale scan.(4)Preparation and verification of rabbit anti-vCXCL-1 polyclonal antibody: the purified protein was used as an immunizing antigen.After being concentrated by ultrafiltration and quantified by BCA,New Zealand white rabbits were immunized with subcutaneous multipoint injection to obtain antiserum.And then purified the antiserum to obtain high-purity polyclonal antibodies.Western blot was used to detect the polyclonal antibodies qualitatively.(5)Application of rabbit anti-vCXCL-1polyclonal antibody: using the obtained polyclonal antibody as the primary antibody to detect the expression of the virus natural protein vCXCL-1 in the cells by Western blot and the localization of the virus natural protein vCXCL-1 in the cells by indirect immunofluorescence.The results obtained by the above method are as follows:(1)Construction of recombinant expression vectors: HCMV UL146 gene was successfully amplified from HCMV BAC Towne genome,the full-length sequence of UL146 was inserted into prokaryotic expression plasmids pET32a(+)between SalI and BamHI restriction sites.The positive clones were confirmed by restriction enzyme digestion and sequencing.(2)Prokaryotic expression of recombinant protein: pET32a(+)-UL146 expression vector was transformed into E.coli BL21(DE3).After the optimization of conditions,the optimal temperature for inducing expression of the recombinant protein in prokaryotic cells was determined as 20℃,the optimal inducer concentration was 0.5 mM,and the optimal induction time was 10 h,and the recombinant protein was highly expressed but in an insoluble form which was comformed by SDS-PAGE and Western blot.(3)Purification of recombinant protein:the higher purity recombinant protein was obtained after purified by affinity chromatography through Ni–NTA Resin,the purity of the recombinant protein was up to 80%,it could be used for animal immunization.(4)Preparation and verification of rabbit anti-vCXCL-1 polyclonal antibody: the pre-immune and immune serum were collected to test the antibodies activities by ELISA.The titer of the polyclonal antibody was 100,000.Western blot analysis showed that anti-vCXCL-1 polyclonal antibody qualitatively detected vCXCL-1 recombinant protein.(5)Application ofrabbit anti-vCXCL-1 polyclonal antibody: Western blot analysis showed that anti-vCXCL-1 polyclonal antibody can detect the expression of the virus natural protein vCXCL-1 in cells;indirect immunofluorescence analysis showed that anti-vCXCL-1 polyclonal antibody can detect the localization of the virus natural protein vCXCL-1 in cells..In summary,this experiment was successfully constructed the pET32a(+)-UL146 recombinant plasmid and transformed into E.coli BL21(DE3).After optimizing the conditions,a high expression recombinant protein in the form of inclusion bodies was successfully obtained.The high purity recombinant protein was obtained by affinity chromatography through Ni–NTA Resin.The purified recombinant protein was used as an antigen for immunizing rabbits to obtain high titer antiserum.The antiserum was purified to obtain high-quality polyclonal antibodies.Qualitative analysis of the polyclonal antibodies showed that anti-vCXCL-1 polyclonal antibody was successfully obtained.And the polyclonal antibody antibody can be used to detect the expression and localization of the viral native protein vCXCL-1 in the cell.