| Objective The recombinant nucleoprotein of measles virus(MV)with good antigenic characteristics was prepared by prokaryotic expression system.As the coating antigen,an indirect enzyme-linked immunosorbent assay(ELISA)method for detection of MV antibody was established and applied to the evaluation of antibody level in the population.Methods The gene sequence of MV nucleoprotein provided by National Center for biotechnology information(NCBI)and national measles laboratory was selected,and 450 nucleotides at the carboxyl end of the protein were translated and analyzed for amino acid sequence consistency,select the target sequence.MV RNA was extracted,and PCR was performed after reverse transcription,After amplification,the full-length sequence of nucleoprotein gene was obtained.The codon of the full-length sequence of nucleoprotein gene was optimized by using related biological software.The target gene was connected to pET-32a(+)expression vector.The recombinant expression plasmid pET-32a(+)-MV-N was constructed and transformed to expression Escherichia coli BL21(DE3).The recombinant protein was induced by Isopropyl β-D-Thiogalactoside(IPTG)and identified by Western Blot with specific antibody.After crude purification by salting out method,the target protein was purified by Ni column affinity chromatography,and was observed by electron microscopy.The indirect ELISA method was established with recombinant nucleoprotein as coating antigen.The working conditions were screened and optimized to determine the operation process of indirect ELISA.The anti-measles virus IgG antibody in 157 sera of healthy children and newborn mothers was detected and compared with the commercial measles virus ELISA IgG antibody detection kit.Results According to the results of sequence consistency analysis,the amino acid sequence with 100%homology was classified into one group,and there were 142 sequences in the group with the largest proportion in the total number of sequences These sequences had a long time span and a wide range of sources.One of them was selected as the template of this study and expressed by prokaryotic expression system.The MV recombinant nucleoprotein was 76KD,which could be recognized by specific polyclonal antibody.After purification,the purity of the protein can reach more than 90%,which can form a typical herring bone like nucleocapsid structure.The results showed that the coefficient of variation of intra and inter assay repeatability of the indirect ELISA was less than 10%.Compared with the results of the commercial kit,the total coincidence rate of serum samples was 95.5%,and the sensitivity and specificity were 94.8%and 98.3%,respectively.There was no significant difference between the two methods(χ2=0.313,P>0.05;χ2=0.000,P>0.05)in the positive rate of measles virus IgG antibody in 85 healthy children of 0～15 years old.The level of measles virus antibody detected by ELISA established in this study showed the same increasing and decreasing trend as that detected by commercial kit in different age groups.The correlation coefficient r of the quantitative results was 0.893(P<0.001),which showed the quantitative potential of the method.Conclusions The recombinant nucleoprotein with good antigenic characteristics was successfully prepared by the prokaryotic expression system.The ELISA method established by the recombinant antigen has good stability,high sensitivity and specificity,and there is no significant difference between the detection results of commercial kit and the ELISA method.It can be used for the seroepidemiological investigation of measles virus and the evaluation of antibody level of the population after vaccination.Figure[thirteen]table[eleven]reference[Forty-one]... |
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