Codon Optimization, Soluble Expression and Purification of PE_PGRS45 Gene from Mycobacterium tuberculosis and Preparation of Its Polyclonal Antibody Protein

Abstract

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti-PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function

“…Using Mtb H37Ra genomic DNA as a template, the target gene was obtained by PCR amplification, and the primers are shown in Additional file 1 : Table S1. We inserted the target gene into the vector pET-28a using the homologous recombination cloning technique [ 27 ]. The obtained 12 recombinant plasmids were confirmed by colony PCR and DNA sequencing.…”

“…His-tagged recombinant proteins were purified by nitrilotriacetic acid (Ni–NTA) agarose resin (CWBIO, Jiangsu, China) according to previous reports [ 28 ] for purification of proteins under soluble conditions. First, the crude supernatant cell extract was filtered through a 0.45 μm filter.…”

Immunological effects of the PE/PPE family proteins of Mycobacterium tuberculosis and related vaccines

“…Recently, Xu et al. purified PPE15 and found that it had strong antigenicity and could react specifically with the serum of patients suffering from TB but not with the serum of patients with pneumonia or healthy adults ( 102 ). Although few studies have been done on PPE15, those results suggest that PPE15 is a promising antigen target for developing vaccine candidates against TB, but the specific immune mechanism merits further exploration.…”