Preparation of human cytomegalovirus latency-associated determinant pUL138 and evaluation of its preliminary use in the serodiagnosis of HCMV infection
1.Department of General Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015; 2.The First Clinical, Wenzhou Medical University, Wenzhou, 325035; 3.Department of Rheumatology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015; 4.Department of Microbiology and Immunology, Institute of Molecular Virology and Immunology, WenzhouMedical University, Wenzhou, 325035; 5.Experimental Center, Wenzhou Medical University, Wenzhou, 325035
CHEN Wenjing1,HUANG Chongan2,CHEN Jing3, et al. Preparation of human cytomegalovirus latency-associated determinant pUL138 and evaluation of its preliminary use in the serodiagnosis of HCMV infection[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2015, 45(10 ): 703-.
Abstract： Objective: To prepare latency-associated protein UL138 of human cytomegalovirus, determine its antigenicity and evaluate its preliminary use as an antigen in the serodiagnosis of HCMV infection. Methods: Bioinformatics methods were used to analyze the transmembrane domain of UL138 protein, and the complete coding region of pUL138’s cytoplasmic domain was optimized by the usage of the favorite codons in E.coli, amplified by PCR and cloned into the expression vector pET21a(+). The constructed pET21a(+)/UL138 plasmid was transformed into E.coli BL21 cells and induced by IPTG. The recombinant protein pUL138 was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis. Then the purified UL138 protein was coated on plates as a diagnosis antigen and an indirect ELISA method was established and optimized to investigate the HCMV-specific IgG. Finally serum samples from 173 healthy individuals were detected by the established method, and Roche’s electrochemiluminescence detection kit was considered as the golden standard to evaluate its significance in the serodiagnosis of HCMV infection. Results: HCMV UL138 protein was successfully expressed in prokaryotic system and highly purified by affinity purification. The positive rate of specific IgG against HCMV was 99.4% by the established ELISA, with no statistically significant difference between the result of pUL138-based ELISA and that of Roche’s detection kit (x2=0.5, P>0.05), and the sensitivity and coincidence rate of the ELISA method were 100.0% and 98.8%. These two results had good coherence (Kappa=0.496, P<0.001). Conclusion: Latency-associated protein UL138 is a potential candidate antigen for HCMV detection and can be useful for development of new techniques for the serodiagnosis of HCMV infection.
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