Extraction, Purification And Serological Application Of P1 Recombination Protein Of Mycoplasma Pneumoniae Purification

Extraction, Purification and serological Application of P1 Recombination Protein of MycoplasmaPneumoniaeIntroductionMycoplasma pneumoniae (Mp) is the causative agent of atypical pneumoniae and is also responsible for other respiratory tract infections in older children and young adults. The traditional methods for the diagnosis of Mp infection is to isolate Mp from clinical samples and Culture it , but Culture requires specialized media, is time-consuming (up to 21 days) and is rarely undertaken in a routine context. At present, the laboratory diagnosis of Mp infection is mainly based on serological tests, such as enzyme-linked im-munosorbent assay(ELISA) and PCR. The conventional serologic tests are considered nonspecific and may cross-react with other Mycoplasma species. In our research, the P1 recombination protein of Mycoplasma pneumoniae was extracted and purified and a serological ELISA with purified P1 recombination protein was established , which may help with the improvement of the sensibility and the specificity of the diagnosis of Mp infection.MethodsThe abstraction and purification of P1 recombination protein: The pGEX-6P-1-P1'recombinant was cultured and Protein expression was induced by IPTG. The bacteria were pelleted by centrifugalization, then the P1 recombination protein was extracted and purified with B-PER GST Fusion Protein Purification Kit. Purified recombinant protein was further subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to check the expression of recombinant proteins and subjected to Western blotting to check its antigenicity.The Mp was pelleted by centrifugalization. Serum samples were collected from adult patients with mycoplasmal pneumonia and then screened for anti-Mycoplasma pneumoniae IgG antibodies through the indirect ELISA ,which was established with P1 recombinant protein and Mp. The optimal concentration of the antigen was definited through the way of block titration. The precision, sensibility and specificity of the antigen was detected by indirect ELISA.ResultsThe SDS-PAGE showed that the molecular weight of P1 recombination protein was about 59kDa. Upon Western blotting, the protein strap can react with the rabbit serum immuned by Mp. We detected 51 clinical samples by ELISA, in which there were 31 positive serum detected by the P1 recombination protein and the positive ratio was 60.78%, and 20 positive serum detected by Mp and the positive ratio was 39.22%. The precision of the positive pooled serum is 5.40%, and the precision of the negative pooled serum is 1.10%.Conclusion1. The P1 recombination protein is more sensitive than intact Mp membrane antigen, so it can be used to detect the infection of Mycoplasma pneumoniae in clinical.2. The P1 recombination protein can be prepared by gene engineering, its cost is very low, and also it standardizes the diagnosis of Mp infection. As a result, the P1 recombination protein can be used to prepare the diagnostic kit for measurement of antibodies to Mp.