Cloning And Expression The Gene Of Momordica Anti-HIV Protein MAP30 In E.coli

MAP30, a ribosome-inactivating proteinⅠ, which was isolated from the seeds and fruits of balsam pear (Momordica charantia L.), was a HIV virus inhibitor and had the properties of anti-virus, anti-tumor and antibiosis. In this study, five MAP30 genes (JX-MAP30, ZY-MAP30, LAN-MAP30, BC-MAP30 and YT-MAP30) were cloned from five balsam pear cultivars. 861bp sequences were obtained by polymerase chain reaction (PCR). Sequence analysis revealed that those genes were the same with the No: AJ294541 gene except BC-MAP30 and YT-MAP30. So, the important study focused on the analysis of the nucleotide sequences and their deduced amino acid sequences of BC-MAP30 and YT-MAP30. Then, the secondary structure of BC-MAP30 and YT-MAP30 proteins were predicted by bioinformatics. On the other hand, the complete encoding sequence YT_f-MAP30 and matured peptide encoding sequence YT_m-MAP30 were both constructed into prokaryotic expression vector. The two recombinated expression plasmids were expressed by optimizing conditition. The induced productions and were analyzed by SDS-PAGE, dissolving nature analysis and immunity characteristic analysis. The results were as following:1. Sequence analysis revealed that there were two different bases in BC-MAP30 and three different bases in YT-MAP30. The open reading frame both encoded 263 amino acid of matured peptide.2.The induced amino acid sequence identity with other cucurbitaceous ribosome-inactivating proteins was up to 68.90%, with TCS was 71.25%, with Gelonin was 71.25%, with Saporin was 66.45%. The amino acid sequences of 90 to 220 were conservative. Secondary structure prediction showed that the random coil was the most, then wereα-helix andβ-pleated sheet. The signal peptide domain was full ofα-helix and random coil. N-terminal domain was full ofβpleated sheet and random coil. Highly helical central domain was full ofα-helix. C-terminal domain was full of random coil.3. The sequence and cloning site of YT_f-MAP30 and YT_m-MAP30 were correct by sequencing. SDS-PAGE of induced protein of pET-YT_m/MAP30-BL21 strain which was induced by IPTG at 30℃for 4h, showed that there was a strengthened protein band in 35 kD protein Marker, and it was dissolved.4. Western blot analysis revealed that the recombinant protein possessed antigenicity, and could specific reaction with rat anti-His tag multiclone antibody.5. Expression of the engineering bacteria pET-YTf/MAP30-BL21 at different time, different temperature and different IPTG inducer level. There was no specific protein band. Those results showed that the signal peptide of YT_f-MAP30 influenced the expression in E. coli BL21 (DE3)PlysS.