The Construcion Of Fusion Vector Of Chicken Mutated IL-18 Gene And NDV HN Gene And Anti-MDCC-MSB-1 Cell Lines In Vitro

Newcastle disease virus(NDV),a kind of oncolytic virus,can replicate and proliferate in the tumor cells but not in the normal cells,thus specifically kill tumor cells.With the in-depth study on anti-tumor mechanism of NDV,it has been thought that NDV hemagglutinin- neuraminidase(HN)protein plays an important role in anti-tumor activity induced by NDV.Interleukin-18(IL-18)has the potential to be used as an immunomodulator in the therapy of malignant tumors and cancer gene therapy.To discuss the effect and mechanism against cancer of ChMIL-18 protein,HN protein and HN-ChMIL18 fusion protein,in this study we constructed expression plasmids(pPICZαA-ChMIL 18,pPICZαA-HN,pPICZαA-HN-ChMIL18).Then the expression and the bioactivities of ChMIL-18 protein,HN protein and HN-ChMIL18 protein were detected in vitro.In this study we used MDCC- MSB-1(MD lymphoblastoid cell line)in vitro to explore the inhibitive effect of ChMIL-18 protein, HN protein and HN-ChMIL18 fusion protein on the growth of MSB-1 cell and its mechanism.Both HN protein and HN-ChMIL18 fusion protein could inhibit the growth of MSB-1 and the effect induced by HN-ChMIL18 fusion protein was stronger than that of HN protein.In addition,the possible mechanisms were further discussed,HN protein can induce apoptosis of tumor cells,and ChMIL-18 can enhance the non-specific anti-tumor immunoresponse.ChMIL18 protein can promote the Proliferation of lymphocytes determined by MTT(3-(4,5- dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromid)method,but no inhibitory action MSB-1.It was built foundation for the effect and mechanism against cancer of HN-ChMIL18 fusion protein in vivo,so it has theoretical as well as practical significance.1.Obtain target DNA and Bio-informatics Analysis 1.1 Obtaining ChMIL18 Gene and Bio-informatics AnalysisA pair of primers specific to chicken mature IL-18 were designed and synthesized according to GenBank,to amplify chicken mature IL-18 cDNA by reverse transcription polymerase chain reaction(RT-PCR)from peripheral blood monocyte of chicken.It was cloned into pMD18-T vector and identified by sequencing.The results showed that this gene contains 507bp encoding 169 amino acids with a predicted molecular weight of 19.5KD and pI of 6.36.Bioinformatics analysis indicated that the sequence contained antigenicity but no glycosylation sites.All the results were consistent with those of ChIL18 gene abord.It is necessary to reconstruct ChMIL18 for a high level expression of ChIL18 in Pichia pastoris.By using long-distance inverse PCR,the codon of Arg(CGA)of 28 site and 37 site of ChIL18 were mutated synonymously to Arg(AGA),which is a bias code of Yeast.The mutated pMD18-T-ChMIL18 was transformed into JM109 strain.The mutated ChIL18 gene clone was obtained successfully by digesting,PCR,sequencing and blasting.1.2 Obtaining HN Gene and Bio-informatics AnalysisA cDNA fragment of hemagglutinin-neuraminidae(HN)was obtained from Newcastle disease virus(NDV)LaSota strain by RT-PCR.A 1.7 Kb fragment was amplified,which conformed to the NDV-HN gene fragment's reported in the Genbank, and cloned into the pMD18-T vector.The recombinant plasmid was proved to be true by enzyme,PCR and sequencing.The sequence and biochemical character are analyzed with Protean.This polypeptide is consist of 577 aa,63.0KD m.w..Its isoelectric point is 7.54. The structure of protein is analyzed which contained 12 Cysteine Residues,5 the potential glycosylation site.Compared HN gene sequence with the corresponding sequence of other strains B1,Clone30,LaSota and so on,the homology of the nucleotide sequencewas 98.8%-99.8%,the homology of the protein sequencewas 98.8%-99.5%1.3 Obtaining HN-ChMIL18 Fusion GeneAccording to MCS of pPICZαA and pBluescript SK+ vector,two pairs of primers with an adapter with were designed.Fusion protein gene of HN and ChMIL18 was separately synthesized and cloned with pMD18-HN,pMD18-ChIL18 as template.DNA fragment was respectively inserted into pMD18-T Vector,and transformed into E.coli JM109,Recombinants were grew in LB culture medium plate and screened.Plasmid DNA was amplified by using PCR and identified by sequening.The results showed that pMD 18-T-HN~f and pMD 18-T-ChMIL 18~f were constructed successfully.pBS SK+ and pMD 18-T-HN~f were digested separately by KpnI and BamHI enzyme, and were linked under T4 DNA Ligase,pBS SK+-HN was constructed and transformed to E.coli JM109,the recombinant was digested by KpnI and BamHI and PCR,the results showed that recombinant plasmid pBS SK+-HN was constructed.And then,pBS SK+-HN and pMD18-T-ChMIL18~f were digested separately by BamHI and NotI enzyme, and were linked under T4 DNA Ligase,pBS SK+-HN-ChMIL18 was constructed and transformed to E.coli JM109,the recombinant was digested by KpnI and NotI and PCR, the results showed that HN-ChMIL18 fusion gene fragment was inserted into pBS SK+.2 Construction of Expression Vector,Inducible Expression and Bioactivities Test of the Recombinant Protein2.1 Construction of pPICZαA-ChMIL18 Vector,Expression and Bioactivities Test of ChMIL18 ProteinSecreted expression vector pPICZαA and pMD18-T-ChMIL18 were digested separately by EcoRI and KpnI enzyme,and were linked under T4 DNA Ligase, pPICZαA-ChMIL18 was constructed and transformed to E.coli JM109,the recombinant was digested by EcoRI and KpnI and PCR,the results showed that recombinant plasmid pPICZαA-ChMIL18 was constructed.ChMIL18 gene fragment was transformed into P.Pastoris X-33 strain by electroporation after pPICZαA-ChMIL18 was lined by SacI enzyme.High-copied transformants were obtained by Zeocin screening.P.pastoris X-33/ pPICZαA-ChMIL18 was expressed under the induction of 1%methanol.SDS-PAGE showed that after being induced with 1%methanol for 4d,the expressed products existed in supernatant and it's molecular weigh was about 23KD,Western-blot showed good antigenicity and specificity of expressed product,lymphocyte transformation test showed ChMIL18 significantly influenced increment reactivity of lymphocyte of chicken. 2.2 Construction of pPICZαA-HN Vector,Expression and Bioactivities Test of HN ProteinSecreted expression vector pPICZαA and pMD 18-T-HN were digested separately by KpnⅠand NotI enzyme,and were linked under T4 DNA Ligase,pPICZαA-HN was constructed and transformed to E.coli JM109,the recombinant was digested by KpnI and NotI and PCR,the results showed that recombinant plasmid pPICZαA-HN was constructed.HN gene fragment was transformed into P.Pastoris X-33 strain by electroporation after pPICZαA-HN was lined by SacI enzyme.High-copied transformants were obtained by Zeocin screening.P.pastoris X-33/pPICZαA-HN was expressed under the induction of 1%methanol.SDS-PAGE and Western-blot showed that after being induced with 1%methanol,the expressed products existed in supematant and it's molecular weigh was about 97KD,has good antigenicity and specificity of expressed product,hemagglutination test showed HN protein could absorb chicken red blood cells(RBC).2.3 Construction of pPICZαA-HN-ChMIL18 Vector,Expression and Bioactivities Test of HN-ChMIL18 Fusion ProteinSecreted expression vector pPICZαA and pBS SK+-HN-ChMIL18 were digested separately by KpnI and NofI enzyme,and were linked under T4 DNA Ligase, pPICZαA-HN-ChMIL18 was constructed and transformed into E.coli JM109,the recombinant was digested by KpnⅠand NotI and PCR,the results showed that recombinant plasmid pPICZαA-HN-ChMIL18 was constructed.HN-ChMIL18 gene fragment was transformed into P.Pastoris X-33 strain by electroporation after pPICZαA- HN-ChMIL18 was lined by SacI enzyme.High-copied transformants were obtained by Zeocin screening.P.pastoris X-33/pPICZαA-HN-ChMIL18 was expressed under the induction of 1%methanol.SDS-PAGE and Western-blot showed that the expressed products existed in supematant and it's molecular weigh was about 103KD,has good antigenicity,lymphocyte transformation test and Hemagglutination test showed HN-ChMIL18 protein significantly influenced increment reactivity of lymphocyte of chicken and absorb chicken red blood cells(RBC).3 Study on anti-MDCC-MSB-1 in vitroIn this study we used MDCC- MSB-1(MD lymphoblastoid cell line)in vitro to explore the inhibitive effect of ChMIL-18 protein,HN protein and HN-ChMIL18 fusion protein on the growth of MSB-1 cell and its mechanism.The inhibitive effect of ChMIL-18 protein,HN protein and HN-ChMIL18 fusion protein on MSB-1 cell by MTT assay,and explored the mechanism through agarose gel electrophoresis and flow cytometry.MTT assay showed that HN protein and HN-ChMIL18 fusion protein inhibited the growth of MSB-I cell,Agarose gel electrophoresis showed that MSB-1 cell treated with HN protein and HN-ChMIL18 fusion protein for 72 hours had DNA ladder.Flow cytometry analysis indicated that apoptotic cells appeared after MSB-1 cell treated with HN protein and HN-ChMIL18 fusion protein for some time.These results suggested that HN protein and HN-ChMIL18 fusion protein inhibited the growth of MSB-1 cell,and the mechanism was relative to inhibiting the synthesis of DNA and inducing apoptosis of some MSB-1,However,ChMIL-18 didn't so.