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| Prokaryotic expression of UL135 protein of human cytomegalovirus and its preparation and identification of polyclonal antibody |
| HU Yingying1, GUO Gangqiang2, YE Sisi2, SUN Xiangwei3, Mao Chenchen3, XUE Xiangyang2, LYU Jieqiang1. |
| 1.Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325027; 2.Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, Wenzhou Medical University, Wenzhou, 325035; 3.Department of General Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015 |
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| Cite this article: |
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HU Yingying,GUO Gangqiang,YE Sisi, et al. Prokaryotic expression of UL135 protein of human cytomegalovirus and its preparation and identification of polyclonal antibody[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2017, 47(5): 336-341.
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Abstract Objective: To construct a prokaryotic expression vector expressing UL135 protein of human cytomegalovirus (HCMV) and prepare a UL135 specific polyclonal antibody. Methods: Bioinformatics methods were used to analyze the transmembrane domain of UL135 protein, and then the cytoplasmic domain synthesized by fully-synthetic gene was cloned into vector pET21a (+) after the prokaryotic codon optimization. The pET21a (+)/UL135 recombinant plasmid was transformed into E.coli BL21 (DE3) and then induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). To prepare specific polyclonal antibody, New Zealand rabbits were immunized by the UL135 recombinant protein which was purified by Ni-NTA affinity chromatography primarily. The sensitivity and the specificity of the antibody were determined by ELISA and Western blot assays. Results: HCMV UL135 protein was successfully expressed in prokaryotic system and highly purified target protein was obtained through Ni-NTA affinity chromatography. New Zealand rabbits could generate specific polyclonal IgG antibody after immunized. ELASE and Western blot analysis determined that the preparative polyclonal IgG antibody could specifically identify the UL135 protein. Conclusion: HCMV UL135 protein is successfully expressed in prokaryotic expression system, and anti-UL135 rabbit polyclonal antibody is successfully prepared for the further research.
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Received: 25 October 2016
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