Construction Of Dual Targeting Anti-breast Cancer Fusion Protein Δ1-9-g129r-t3 Prokaryotic Expression Plasmid And Inducible Expression

Objective Using recombinant DNA technology, the human prolactin receptor(hPRLR)antagonistΔ1-9-G129R-hPRL with specific inhibition of breast cancer cell proliferation and the active subfragment of tumstatin T3 peptide (69-88aa) with specific anti-angiogenesis activity were connected together to construct a new dual targeting anti-breast cancer fusion proteinΔ1-9-G129R-T3 prokaryotic expression vector.Transform it into a prokaryotic expression host and then carry out target protein induction of expression, identification , to lay the foundation for further functional testing of the fusion protein.Methods First of all ,the gene fragmentsΔ1-9-G129R-hPRL with restriction enzyme NdeI and XhoI,and with NdeI and BamHIrecognition sequence at each end were amplified by method of PCR from the template of plasmid pET22b(+) G129R-G129R. Extract the DNA fragments by agarose gel electrophoresis. According to the cDNA sequence of T3, three strips of complementary primers were designed and synthesized, the DNA fragments of T3 with restriction enzyme NdeI and XhoI,and with BamHIand XhoI recognition sequence were amplified using overlap extension PCR technique.Recovered DNA by polyacrylamide gel electrophoresis.To make use of the complementary of BamHI recognition sequence in the 3' end ofΔ1-9-G129R-hPRL and the 5' end of T3,the purifiedΔ1-9-G129R-hPRL and T3 mixed as template, fusion gene fragmentΔ1-9-G129R-T3 with restriction enzyme NdeI and XhoI recognition sequence was PCR amplified using the upstream primer ofΔ1-9-G129R-hPRL and the downstream primer of T3. The purifiedΔ1-9-G129R-T3,Δ1-9-G129R and T3 were respectively cloned into pMD18-T vector by T-A cloning and then transformed into E. coli DH5α, the positive clones were picked by the blue-white selection, plasmids were extracted after clonal expansion and identified by restriction enzyme digestion with NdeI and XhoI,and then recovered the DNA fragments.The gene fragments after restriction enzyme digestion and purification were respectively inserted into the NdeI and XhoI cloning site of pET22b(+),to construct the prokaryotic expression vector pET22b-Δ1-9-G129R-T3, pET22b-Δ1-9-G129R and pET22b-T3.Transformed these three recombinant expression plasmids which have right DNA sequencing into prokaryotic expression host E. coli BL21 (DE3), induced expression of protein with IPTG and the collected cell, isolated precipitation and supernatant by high speed centrifugalization after ultrasonic fragmentation cell. Finally the expression of fusion protein were detected by SDS-PAGE and Western blot.Results Detected with agarose gel electrophoresis,target we got the target gene fragmentsΔ1-9-G129R-T3 (650 bp),Δ1-9-G129R-hPRL (586bp) and T3 (72bp) by PCR amplification method. Inserted the purified target gene double digested with restriction enzyme NdeI and XhoI into pET22b(+) vector double digested with the same enzyme to construct three prokaryotic expression vectors pET22b-1-9-G129R-T3,pET22b-Δ1-9-G129R,pET22b-T3.Recombinant expression plasmid were double digested, the results show that the size of digested fragments conformed to the inserted gene fragment .DNA sequencing indicated the cDNA are same with the cDNA sequences registered in GenBank except two silent mutations. Transformed these recombinant expression plasmid into BL21 (DE3) and induced expression of protein with IPTG .SDS-PAGE electrophoresis confirmed that there was an apparent new protein stripe in the precipitation with ultrasonic treatment, and the size of which conformed to the expected molecular weight (about 24.7 kD, 22.2 kD, 2.4 kD). Target protein mainly exists in the form of insoluble inclusion bodies, located in the cytoplasm. Western blot analysis showed that the blot of inducible expression of the bacterial protein appeared at about 24 kD and 22kD, indicated that foreign gene has been successfully expressed in E. coli.Conclusion The prokaryotic expression plasmid of fusion proteinΔ1-9-G129R-T3 was constructed successfully and can be expressed efficiently in prokaryotic expression host E.coli BL21 (DE3) cells ,which will help us to further study the function of fusion protein.