Cloning And Expression Of Gene Encoding Chlamydophila Pneumoniae Major Outer Membrane Protein And The Antigenicity Analysis Of The Protein

Chlamydophila pneumoniae (Cpn) is an obligate intracellular bacterium, was first isolated from the conjunctiva of a Taiwanese child in 1965, was named as Chlamydia pneumoniae in 1989, and was renamed as Chlamydophila pneumoniae in 2004. Similar to other chlamydia species, it is known that the infection of cells with Cpn is initiated by an environmentally resistant electron-dense form termed the elementary body (EB). The transition from EB to the metabolically active replication cell, called the reticulate body (RB), begins within the first few hours after infection. The RBs multiply by binary fission until the late phase of infection and begin to convert back to Ebs. Cpn is responsible for sinusitis, bronchitis, and 10-15% of community acquired pneumonia cases in the worldwide. By age 20, about 50% of the population exhibits evidence of past infection by C. pneumoniae and re-infection is common throughout the life. Currently Cpn has attracted increasing interest because it is associated with an array of chronic human diseases, such as atherosclerosis, endocarditis, multiple sclerosis and asthma .Major outer membrane protein(MOMP) is one of chlamydia characteristic antigens,accounting for over 60% of chlamydial outer membrane proteins and presenting both in EB and RB. OmpA gene encoding Cpn MOMP reveals that it consists of a 1,167bp open reading frame with an inferred 39,344-dalton mature protein of 366 amino acids plus a 23-amino-acid leader sequence. This sequence shares 68 and 71% DNA sequence homology to the Chlamydia trachomatis serovar L2 and Chlamydophila psittaci ovine abortion agent MOMP genes, respectively. Sequences alignment revealed that the OmpA gene is conserved. MOMP played important role in Cpn infecton. A great deal of studies indicated that MOMP could be recognized by sera from patients with Cpn infections in Westen Blot. Due to great difficulties with extracting and purifing MOMP from natural Cpn in commercial scale,researchers attempted to obotian recombinant Cpn MOMP(rCpn-MOMP) by gene engineering.In the study, primers was designed according to Cpn OmpA gene sequence(Gene Bank accession no. M69230) , and the sequence of Cpn OmpA except signal was amplified from the purified Cpn genomic DNA of ATCC VR1360 by PCR. 1% agarose electrophoresis analysis showed that the amplified product was 1100 bp. Amplified product was inserted pMD18T vector to construct clone plasmid pMD18T-MOMP. JM109 was transformed with the clone plasmid and the clone strain was confirmed by PCR. After pMD18T-MOMP and pET32a vector was treated with NcoⅠand SalⅠ, expression plasmid pET32a-MOMP was construced by ligation. BL21(DE3) was transformed with the plasmid to obtain expression engineered strain. By identification, the inserted sequence was correct, and due to syncretizing thioredoxin protein, the molecular weight of rCpn-MOMP was about 56KDa. The inserted sequence was 1738bp, an the open reading frame encodeing 527 aminos was from 64bp to 1644bp. Aligned by NCBI BLAST, the sequense identities was 99%. The open reading frame was right to cofirm rCpn-MOMP primary structure right. The rCpn-MOMP expression level was as high as 53% in inducing 4.5h in 28℃, IPTG 1mM , and purification of rCpn-MOMP was over 90% and 1000ml LB culture media could pruduce 350mg purified protein.The antigenicity of rCpn-MOMP was cofirmed by WB. RCpn-MOMP could be recongized with murine anti-Cpn antibodys. Simultaneously rCpn-MOMP could react with Sera from patients with Cpn antibodys .