Codon Optimization Technology And Its Application In The Immunoassay Of Mycoplasma Pneumoniae

Mycoplasma pneumoniae (Mp) is a primary pathogenic microorganism of atypicalpneumonia of children and adolescents. Approximately21.1million new cases(children under age5) of clinical pneumonia appears in China each year(0.22times/person-years), and there is an increasing trend every year. The symptoms of Mppneumonia infection are similar to Streptococcus pneumoniae, HaemopHilusinfluenzae, Chlamydia pneumonia. The treatment of Mp infection is mainly focus onmacrolides, quinolones, Mp is not sensitive with beta-Inner pHthalocyanine amineantibiotics such as penicillin, cepHalosporins. Therefore, the development of sensitive,specific, convenient and early diagnostic reagents will benefit the clinic treatment ofMycoplasma pneumoniae infection.However, antigens used in immunoassay were prepared mainly by extraction andpurification of membrane proteins of Mp pathogens, which caused low specificity forits cross-reaction with Mycroplasma. genitalium (Mg). Cell surface adhesion proteinP1, P30, P116of Mp has been reported having immunogenicity and antigenicity,which is important for the development of Mp immunoassay. However, the codonsystem of Mp is special. The universal termination codon UGA encodes tryptopHan inMp, while in the E. coli expression system, it is a stop codon. Therefore,the wild Mpadhesion protein gene is difficult to express efficiently in E. coli and other expressionsystem. In this study, codon-optimized Mp gene was obtained and effectivelyexpressed in E. coli. The results are summarized as follows:First of all, entire sequences of the P1, P30, P116protein of Mp representativestrains were obtained from Genbank. By predicting and blasting with the top10sequences with the highest similarity, the final advantage epitope regions weredetermined before codon optimization. Mycoplasma pneumoniae P1protein antigenicepitopes are mainly located in the1154-1526aa, to obtain the P1optimize codongene,39rare codons and two stop codon were mutated; antigenic epitopes of P30located in the23-274aa and39rare codons were optimized and mutated; advantage epitope of P116is located in the761-960aa and14rare codons were optimized.Bioinformatics software Biosun are used to design for three optimized segmentof the target gene primers, each full-length target gene is divided into3to6fragmentsof approximately200bp, every fragment was amplified by three round PCR, threepairs of upstream and downstream primers were designed for each round. Theoverlaps between primers were12bp. The length of every primer was28bp to58bpand78primers were finally designed for the gene synthesis.After optimizing of the vectors, vector pGEX-4T-2and pBVIL1-His were choseto express the target gene and the plasmids were transferred into recipient strainHB101for efficient expression.. Expressed in E. coli, GST-P1, IL1-P30, P116-the IL1fusion protein molecular weight65.9kDa,46.5kDa and39.4kDa. Using GST columnand Ni column, the purity of P30, P116fusion protein is lager than90%.Finally, the fusion proteins were used as coating antigen for indirect ELISA assaywith serum samples of Mp infected patients and normal person. The results show thatthe three purified fusion antigen GST-P1, IL1-P30, IL1-P116display specific immunereactivity with MP serum. Compare with Japan's Fuji commercial kits Race MusicDiaz-Michael II (passive agglutination), the P1obtained in this research fusionantigen with commercial kits have the same detection rate of78.26%(18/23), theconsistency is of up to69.56%(16/23). That means the P1antigen displayed potentialdiagnostic value.In summary, this study screened advantage epitopes of three important adhesiongenes of Mp though bioinformatics method. The cross-reactivity with pathogens suchas Mycoplasma genitalium was avoided by this method. We designed and optimizedthe encoding DNA sequence for the advantage epitope of adhesion protein P1, P30,P116and overcome the codon bias differences between the the Mp systems with E.coli system, the recombinant Mp antigen acquired will benefit the development of Mpdiagnostics and display potential clinic application.