Expression,purification and bioinformatics analysis of PE＿PGRS35 protein of Mycobacterium tuberculosis H37Rv strain / 中国生物制品学杂志

ABSTRACT

@#Abstract： Objective To clone PE_PGRS35 gene of Mycobacterium tuberculosis（MTB），construct recombinant vector pET28a⁃PE_PGRS35，express and purify the PE_PGRS35 protein of MTB H37Rv heterologously，and explore a new target against MTB after bioinformatics analysis. Methods The PE_PGRS35 coding gene was amplified by PCR and used to construct the expression vector pET28a⁃PE_PGRS35 by recombinant cloning technology，which was transformed to E. coli BL21（DE3）after successful sequencing and induced by using IPTG. The obtained PE_PGRS35 protein was purified by Ni column affinity chromatography and analyzed by bioinformatics. Results The pET28a⁃PE_PGRS35 prokaryotic expression vector was constructed correctly as identified by sequencing. The PE_PGRS35 protein was mainly expressed in the form of inclusion bodies，with a relative molecular mass of about 53 000 and a purity of 90%. Bioinformatics analysis showed that PE_PGRS35 protein was an acid⁃labile protein，with main secondary structure of β⁃sheet and random coil，and no transme⁃ mbrane region，which was presumed to be an extramembrane protein with 39 phosphorylation sites and two conserved domains. Total 10 proteins，including Rv1769，PPE8，PPE64，PPE54，PPE24，PPE16，PPE35，PPE6，PPE28 and PE2， interacted with PE_PGRS35 protein. Conclusion PE_PGRS35 protein with high purity was successfully obtained，which provided a reference for the further development of new targets for drugs against MTB.