Cloning and Expression of Truncated Chlamydial Major Outer Membrane Protein in E.coli: A Miniature Step Forward

Abstract

Administration of Prophylactic vaccines still is the cutting-edge of prevention, control and elimination of infections. Among the common causes of sexually transmitted diseases, chlamydia trachomatis is an important socioeconomic burden worldwide which impairs human genital tract function, resulting in severe complications like urethritis, pelvic inflammatory disease (PID), getting ease of HIV transmission, playing cofactor role in human papilloma virus (HPV)-induced cervical neoplasia, and unfortunately infertility. Because of all difficulties, vaccination is considered to be the most economical and reliable way to escape from unrestrained exacerbation states of infection than any other prevalence program. But despite much advancement in the field of veterinary, the dream has yet to be realized. Among a number of potential candidates, the major outer membrane protein (MOMP) is a vanguard of subunit vaccines. In this attempt, MOMP217 gene fragment cloned in PET28b+ and expressed in E.coli. The protein expression confirmed by SDS-PAGE. These findings demonstrate that, considering the antigenicity prediction and due to the existence of potential epitopic region, rMOMP217 could be a potential capable peptide which can represent as an alternative to whole MOMP. Journal of Vaccines & Vaccination Cloning of PET-MOMP217PCR was performed to amplify the MOMP217 containing epitopic regions with specific primers. Qiagen gel extraction kit (Qiagen, Hilden, Germany) was used to purify PCR amplified product and plasmid DNA. Purified plasmid and amplified insert were double digested with BamHI and HindIII (Fermentase). The digested products were purified and ligated into linearised and purified vector withT4 DNA ligase enzyme(Fermentase), then ligation mixture were transformed into E.coli DH5α cloning host. As well, Recombinant clones were confirmed by colony PCR and double digestion. The confirmed purified DH5α/ pET-MOMP217 positive colony were transformed and cloned into E.coli BL21 (DE3) expression host and used for protein expression purpose with IPTG. Protein expressionE.coli BL2 (DE3) with T7 promoter, harboring the PET-MOMP217 (24kDa) positive colony were grown in 100 ml LB broth at 37°C including 100 µg/ml kanamycin (Sigma) to achieve an optical density (OD) of 0.7 (stationary growth phase) at 600 nm at this time, 1 mM isopropyl-ß-D-thiogalactoside (IPTG; Invitrogen) were added to 50 ml LB broth and the other same amount were considered as uninduced (-IPTG) control. Incubation was continued at 37°C. The samples from induced and uninduced culture were collected within 2, 4, 6 hours and overnight after induction. Incubation temperature in the case of overnight was reduced to 25°C. Samples were accumulated, centrifuged at 6000 rpm for 5 min. Harvested cells were resuspending with 2x loading protein buffer containing 2-ME, boiled and subjected to SDS-PAGE analysis. Total protein expression trend were evaluated by 10% SDS-PAGE stained with Comassie Brilliant Blue. SDS-PAGE were shown to have one band corresponded t...