Expression Of ScFv-LDM And RGD-LDM Fusion Protein In Streptomyces Strain & The Application Of Multi-gene Combination Interference For Cancer Therapy

Part 1. Expression of scFv-LDM and RGD-LDM fusion protein in Streptomyces strainLidamycin (LDM), a novel antitumor antibiotic, is produced by Streptomyces globisporus C-1027, is. It is composed of an apoprotein (LDP) non-convalently linked with a chromophore (LDC). Chromophore possesses great cytotoxicity but is unstable; apoprotein is composed of 110 amino acids and acts as a stabilizer to maintain the unstable chromophore. The lidamycin biosynthesis gene cluster has 56 open reading frames (ORFs) within about 75 kb DNA. The apoprotein coding gene cagA is between OREA1 and ORFA4.The immunoconjugates of monoclonal antibody and chemotherapeutants is an important part of cancer target therapeutic. The given chemotherapeutants can conjugate different monoclonal antibody to produce a series of antibody drugs. Anti-type IV collagenase monoclonal antibody and its single-chain Fv fragment (scFv) can effectively inhibit the growth of tumor cells. LDM-antibody conjugates show exceptional promise as "warhead" drug candidates, and have high potency against tumors with low toxicity. We have successfully utilized E.coli expressing system to prepare a scFv-LDP. The scFv-LDP can be reconstituted with LDC to form active scFv-LDM fusion protein. In this study, we try to change the coding gene of S. globisporus C-1027 by homologous recombination technique to construct a recombinant Streptomyces strain that can stably express and excrete scFv-LDM. The experiment project is to replace the cagA sequence with cagA-scFv sequence and by homologous recombination technique.Taking total DNA of S. globisporus C-1027 as template, E arm and G arm was obtained by PCR. E arm located at left side of cagA gene and G arm right. Taking plasmid pEFL that containing scFv coding gene sequence as template, scFv coding gene was obtained. Taking plasmid pUO9090 as template, the apramycin resistance gene was obtained. The above E arm, G arm, scFv coding gene and apramycin resistance gene were linked to plasmid pBS03 which contain a thiostrepton resistance gene, finally plasmid pBS-scFv was successfully constructed. In order to keep the activities of LDP and scFv protein, a spacer (GGGGS) was used to link them. The plasmid pBS-scFv was transformed into E.coli ET12567, and then Conjugal Transfer was carried out between E.coli ET12567 and S. globisporus C-1027 to form a recombinant strain S. globisponis C-1027-scFv.The recombinant strain S. globisponis C-1027-scFv was confirmed by PCR and Southern blotting. The result show that recombinant strain S. globisponis C-1027-scFv was successfully constructed. The LDP coding gene cagA has been replaced by fusion protein coding gene cagA-scFv.Because LDM has parallel activity of anti-tumor and anti-bacteria, the anti-bacteria activity was taken to replace the anti-tumor activity. The fermentation broth of S. globisponis C-1027-scFv showed anti-bacteria activity. The anti-bacteria activity was appeared after 60h fermentation, then increased up to 80h, and disappeared at 108h fermentation.The result of SDS-PAGE showed that the recombinant strain did not produce LDM band. However, it produced a new protein band with 38kD molecular weight at corresponding location of scFv-LDM fusion protein. This new protein band did not be produced by S. globisponis C-1027. The results suggested that scFv-LDM fusion protein was produced by S. globisponis C-1027-scFv.ELISA analysis showed that the fermentation broth combined to type IV collagenase with high affinity. Western blot analysis further indicated that recombinant strain could successfully express scFv-LDM fusion protein.The peptides containing RGD (Arg-Gly-Asp) motif have high-binding affinity to theα_Vβ₃ integrins in tumors. Theα_vβ₃ integrins have been found to be specifically associated with angiogenesis in tumors. To construct the LDM fusion protein expression technical platform, preparing RGD-LDM fusion protein was also carried out. DNA sequence of RGD peptide is synthesized based on the streptomyces codon. RGD peptide has been linked to the C end of LDP. The recombinant strain S. globisponis C1027-tenp has been identified by PCR and southern blotting. By antibacterial ring test, the recombinant strain S. globisponis C1027-tenp also show anti-bacteria activity.Take together, we successfully constructed recombinant strain S. globisponis C-1027-scFv and S. globisponis C-1027-tenp. Recombinant strain S. globisponis C-1027-scFv can produce and excrete scFv-LDM. It is necessary for the future experiments to increase the producing yield ofscFv-LDM. Part 2. The application of multi-gene combination interference for cancer therapyRNA interference is a potential cancer gene therapy strategy. There are two kinds of RNA interfering agents used for therapy: chemically synthesized small interfering RNA (siRNA) and short hair RNA (shRNA) expression vector. To realize multi-gene combination interference, a plasmid pCSH1 with several shRNA transcription units is constructed.Oncogene ras and myc are frequently mutated or overexpressed in most tumor cells. The expression of N-ras and c-Myc gene has previously been shown to be elevated and deregulated in the human hepatoma cell line HepG2. In this study, we constructed a plasmid pCSH1-NM, containing two shRNA expression cassettes against N-ras and c-Myc, respectively.RT-PCR and western results showed that pCSH1-NM synchronously inhibited N-ras and c-Myc gene expression in HepG2 cells. Growth inhibition assay showed that pCSH1-NM resulted in stronger growth inhibition compared with pCSH1-Nras or pCSH1-cMyc, indicating that dual inhibitions of N-ras and c-Myc were more effective.Hochest stain showed that the ratio of apoptosis in pCSH1-NM-treated cells was higher than that in pCSH1-Nras or pCSH1-cMyc treated cells. Moreover, the cell density of control and pCSH1-Mock is higher than that of pCSH1-NM, pCSH1-Nras and pCSH1-cMyc. The results suggest that combination inhibition of N-ras and c-Myc might enhance apoptosis.Microarray analysis showed that the number of gene expression difference (up-regulation expression or down-regulation) induced by pCSH1-Nras was higher compared with pCSH1-cMyc. However, an intervenient number was induced by pCSH1-NM. Three pro-apoptosis genes (TSC22, GMF, NRIF3) were synchronously expressed in pCSH1-cMyc or pCSH1-NM treated cells, indicating that inhibiting N-ras and c-Myc induce apoptosis in HepG2 cells.In summary, oncogene N-ras and c-Myc expressed abnormally in HepG2 cells can be inhibited by RNA interference. Simultaneous interferences of N-ras and c-Myc showed more effective than interference of N-ras or c-Myc gene alone. Multiple-target gene therapy by RNA interference might be potential strategy for cancer therapy.