Construction And Bioactivity Analyzing Of Recombinant Functional Fragments In MAP30

MAP30(Momordica anti-HIV protein of 30kDa)is a 30kDa single-stranded, type-Ⅰribosome inactivating protein, reported to possess anti-HIV, anti-tumor and antimicrobial activity. It's cytotoxicity is specific to tumor-transformed or viral-infected cells, while showing no adverse effects on normal cells. This specific character making it a candidate for clinical applications. It was found that, after proteolytic digesting, some fragments of MAP30 also had the same anti-HIV, anti-tumor and antimicrobial activity as MAP30, while without ribosome inactivation. It showed that all these activities of MAP30 may be controlled by different positions of the protein. Thus, to clone the specific bioactive fragments(gene segments) is good for obtaining low cytotoxic and high bioactive genetic product.Four functional fragments in MAP30 were amplified by PCR. After sequencing analysis, they were subcloned into the vector pET22b to construct the expression vectors: pET22b- M1,pET22b-M2,pET22b-M3,pET22b-M4. The vectors were transformed into E.coli BL21(DE3)by calcium chloride transformation method, and the recombinants were identified by PCR anlysis. All of the four recombinant functional fragments were expressed in inclusion body with 1mM IPTG induction in E.coli , and the amount reached highest at 28℃,1mM IPTG , 5 hours of induction. The cytotoxicity assay of the four recombinant functional fragments also showed that they exhibited dose-dependent inhibition to wish, NFS-60 and S-180 lines , and obviously to NFS-60. The dose required for 50% inhibition (ID50) of M1, M2, M3, M4 and MAP30 were 2.51μg/ml, 2.42μg/ml, 1.89μg/ml, 1.90μg/ml and 40.0μg/ml, respectively. However, little cytotoxic effect on HL-70 were found under the same assay condition. Among these proteins, M3 has the highest anti-tumor activity, also It has the lowest cytotoxicity to normal cell.The results demonstrate that the recombinant M3 expressed in E.coli is active to tumor cells, and the activity is better than MAP30, while the cytotoxicity is lower. The protein M3 isn't consist of the ribosome inactive sequence:C-terminal, so the cytotoxicity is lower. The length of M3 is almost 100aa shorter than MAP30, so we predict it will easier to be ingested, and can degrade antigen-antibody reaction. These findings may offer a significant perspective in both clinical application and market application.