The Effect of Recombinant Fusion Peptide Capsular Biosynthesizing Enzymes A, C Streptococcus agalactiae and Anti-Cancer Peptide against Cell Line Hela by Real-time RT- PCR and Flow cytometry

Abstract

There have been numerous reports of the effect of the Streptococcus agalactiae peptide and its capsule products on cervical cancer. This study aimed to investigate the inhibitory effect of the recombinant anti-cancer protein CpsA-CpsC-L-ACAN on the HeLa cell. The CpsA-CpsC-L-ACAN sequence construction was obtained from NCBI. The Structure of CpsA-CpsC-L-ACAN was examined using various bioinformatics software programs. After creating and cloning into the expression vector pET-22b (+), CpsA-CpsC-L-ACAN was transferred to E. coli BL21(DE3). Nickel column chromatography and Western blotting were used to perform purification and confirmation of CpsA-CpsC-L-ACAN. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) approach was utilized for investigating the cell-killing effect of different concentrations of CpsA-CpsC-L-ACAN against the HeLa cell. Moreover, using Real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), the expression of apoptotic genes, like Bcl-2, caspase-3, and Bax before and after exposure to CpsA-CpsC-L-ACAN was measured. Then, CpsA-CpsC-L-ACAN impact on HeLa cells was examined by flow cytometry. The antibacterial properties of the CpsA-CpsC-L-ACAN were evaluated by the minimum inhibitory concentration (MIC) test and the disk diffusion test. CpsA-CpsC-L-ACAN at a concentration of 64µg/ml killed 50% of cancer cells in 24 hours and after the treatment of Hela cells with CpsA-CpsC-L-ACAN protein, the expression of apoptosis genes, caspase-3 and Bax increased 16 and 6 times, respectively. Also, the expression of bcl-2 by 0.176 times decreased. According to the results of the flow cytometry test, after treatment with CpsA-CpsC-L-ACAN, the cancer cell population transitioned from the living phase to the apoptotic phase.