Construction Of High Efficient Engineering Bacteria Of Vascular Basement Membrane-derived Multifunctional Peptide And Therapeutic Trial Of Human Lung Cancer Xenograft In Nude Mice

Background:Used the strategy of integrative suppression tumor angiogenesis and suppression tumor cell multiplications have recently obtained a novel hybride protein namely Vascular basement membrane-derived multifunctional peptide(VBMDMP) by ligation of Tumstatin N 74-98 amino acid function peptide and 197-215 amino acid function peptide via human IgG3 upper hinge region: This study is based on this, and constructed dissolubility fusion protein prokaryotic expression plasmid pET SUM0-4VBMDMP with high abundance expression, and transformed into Ecoli. BL21 (ED3) to obtain high performance expression of recombinant VBMDMP. Therapeutic trial of human lung cancer xenograft in nude mice was used to evaluate the anti-tumor efficiency of recombinant VBMDMP. It is the foundation for industrialization of recombinant VBMDMP. Methods:The pUC19-VBMDMP and pUC19 were digested with Hind III and Kpn I respectively, and obtained VBMDMP cDNA and pUC 19 cDNA, we ligated the two fragments, get new unimer pUC19-VBMDMP. After identification by restriction endonuclease digestion and sequencing, the new unimer pUC19-VBMDMP was digested with BamH I and EcoR I , and with Bel I and EcoR I ,and VBMDMP cDNA and vector cDNA wre obtained, respectively. The two fragments of cDNA were ligated, and we obtained pUC19-2VBMDMP . Repeated above step ,we can obtain pUC19-4VBMDMP. Meanwhile, pUC19-2VBMDMP and pUC19-4VBMDMP were confirmed by restriction endonuclease digestion and sequencing respectively. TakenpUC19-4VBMDMP as template, T/A clone method was used to constructe recombinant plasmid pET SUM0-4VBMDMP. Recombinant plasmid pET SUM0-4VBMDMP was transformed into Ecoli. BL21(ED3) for obtaining recombinant 4VBMDMP fusion protein. At the same time, we identified recombinant 4VBMDMP using western blotting with polyclonal antibody against tumstatin N 197-215 amino acid function peptide. BCA method was used to memsure the transformed Ecoli.BLZl (ED3) lysate and nickel affinity chromatograph elution protein concentration, and estimate efficiency of producting recombinant VBMDMP by the transformed Ecoli.BL21 (ED3). Therapeutic trial of human lung cancer xenograft in nude mice was used to investigate the anti-tumor activity of recombinant VBMDMP. Results:(1) Unimer, dimmer and tetramer gene of recombinant VBMDMP gained from polymerase chain reaction production of pUC19-VBMDMP, pUC19-2VBMDMP, pUC19-4VBMDMP and the plasmids. The sequences of those recombinant genes were confirmed to consistent with our designed sequence by restriction endonuclease digestion and sequeciong.(2) The 43KD targeting bar was showed by SDS-PAGE and western bloting in bocterium lysate and nickel affinity chromatograph elution of Ecoli. BL21 (ED3) transformed pET SUM0-4VBMDMP and induced by IPTG for 4h. It is recombinant 4VBMDMP.(3) Lysate of pET SUM0-4VBMDMP transformed into Ecoli.B121(ED3) contains protein 5.45mg/ml, while elution by nickel affinity chromatograph contains 2.56 mg/ml. Targeting protein expression of the transformed bacteria is 47%. Producetive effciency of the transformed bacteria is 9.8 times more than that of Ecoli. JM109 by transformation with pGEX-4T-l-VBMDMP.(4) The inhibitory rate of xenograft growth was 42%, 74% treated with recombinant VBMDMP at lmg.kg"1 and 5 mg.kg"1, respectively. The inhibitory rate of xenograft weigh was 42 %, 77% treated with recombinant VBMDMP at lmg.kg"1 and 5mg.kg~\ respectively. These result indicated recombinant VBMDMP possess significant inhibitory growth effect of human lung cancer xenograft in nude mice.