Construction Of Antitumor Peptide Targeted Bacteria And Its Antitumor Activity

In recent years,more and more attentions have been paid to exploit anti-tumor product resources and construct tumor-targeting engineering bacteria,and to innovate new approaches for treating tumor using targeting engineering bacteria.In our study,the nucleotide sequences coding for T7 peptide,19 peptide,antimicrobial peptide AP1 derived from tumstatin and Janthinobacterium Lividum were synthesized.In addition,the fusion gene composed of these three genes was also constructed.The protein expression vectors were constructed by cloning the coding sequences of T7 peptide,19 peptide,AP1 peptide and fusion protein into the expression plasmids respectively.Soluble expression of T7 peptide,19 peptide and AP1 peptide in E.coli BL(DE3)was achieved on expression vectors pSmart ? and pSmart ? with different fusion tags.T7 peptide,19 peptide and antimicrobial peptide AP1 were purified by Ni column and their antitumor activity in vitro against human cervical cancer HeLa cells,mouse melanoma B16 cells and human hepatoma 7721 cells.The results showed that T7 peptide had the highest activity on HeLa cells.Especially,T7 peptide had the highest and most stable inhibitory effect.A nude mouse model of HeLa cervical cancer was established by subcutaneous injection.The engineered strain EcN-Lux labeled with luciferase was transported to the hypoxic area of HeLa cervical cancer tissue by intravenous injection.The tracer was detected by IVIS system.The results showed that the EcN-Lux was concentrated in cervical cancer tissues,but was completely eliminated from liver,spleen and kidney tissues of nude mice.The targeting therapy of EcN-Lux had no obvious effect on body weight and main immune organs of nude mice,this result implies the negligible side effect on the liver,kidney and spleen.In order to study the antitumor activity of engineering bacterium expressing T7 peptide in vivo,EcN was used as T7 peptide targeting carrier.The T7 peptide gene sequence was successfully controlled under hypoxic promoter Pvhb by overlapping PCR,producing the engineering strain of EcNT7.The anti-tumor mechanism of EcNT7 engineering strain was studied through nude mice bearing tumor.Immunohistochemistry and immunofluorescence analyses detected a large number of infiltrating inflammatory cells in the cervical cancer tissues of nude mice treated with EcNT7 engineering bacteria.EcNT7 engineering bacteria play an important role in promoting cell apoptosis and inhibiting cell proliferation,showing that T7 peptide has high anti-tumor activity in vivo,and can significantly inhibit the proliferation of HeLa cervical cancer in nude mice.