Prokaryotic expression and preliminary application of P30 adhesin of Mycoplasma pneumoniae.

Objective To construct a prokaryotic expression vector for the P30 adhesin gene of Mycoplasma pneumoniae(MP),develop a dolt-ELISA using the purified recombinant P30(rP30)protein as antigen and evaluate the significance of rP30 indiagnosis of MP infection.Methods P30 gene was amplified by PCR using the genomic DNA of MP as a template and inserted intovector pET-32a(+).The constructed recombinant plasmid pET-32a(+)/ P30 was transformed to E.coli BL21(DE3)for expressionunder induction of IPTG.The expressed protein was determined for reactogenicity by Western blot.An indirect dolt-ELISA methodwas developed using the purified rP30 protein as antigen and used for detection of 40 serum samples from suspected children with MPinfection.Results The expressed rP30 protein with a relative molecular mass of about 52 000 contained about 13% of total somaticprotein,mainly existed in a form of inclusion body,and reached a purity of about 93% after purification by Ni2+-NTA chromatography.Western blot showed specific reaction of rP30 protein with the sera of children with MP infection.The sensitivity,specificity and ac-curacy of dolt-ELISA developed using rP30 as antigen were 95.45%,72.22% and 85% respectively.Conclusion Recombinant P30protein was successfully expressed in E.coli,which provided a novel tool for rapid diagnosis of MP infection and laid a foundation offurther study on function of P30 as well as the role of P30 in onset mechanism of MP infection.