Construction Of A Recombinant Adenovirus With Hemagglutinin-neuraminidase (HN) Gene Which Is Co-regulated By MTERT And MTyr Promoters And Its Research On Targeting Therapy Of Melanoma Cell Line B16

Melanoma is a kind of common skin malignant tumor covers 1%³%of all malignant tumors.Melanoma not only has a high degree of malignant,and its?metastasis always occurs early and widely,but also has strong resistance to conventional treatment,which is tricky on clinical treatment of malignant tumor.Because of melanoma often occurs in the skin,which is easy to be observed and suitable for gene therapy research.With the development of molecular biology,immunology,pathology and related disciplines,gene therapy has shown a lot of great advantages,and melanoma has gradually become the hot spot of oncology research.Currently,adenovirus?adenovirus,Ad?vector therapy is one of the most promising methods of gene transfer in gene therapy,which can effectively transfer exogenous gene into various target cells or tissues.The benefits of adenovirus vector are as following,it is easy to construct and operate,it has a broad spectrum of host range,and it can cause strong infection of differentiated cells by different approaches into different tissues.In this research,we chose hemagglutinin-neuraminidase?HN?gene comes from Newcastle diseases virus?NDV?as the therapeutic gene for the treatment of melanoma.Lots of research results have shown that the HN protein is the main component of NDV antitumor effect.HN protein not only can mediate virus adsorption with fusion protein on the surface of tumor cells,and facilitate virus to invade tumor cells,but also has neuraminidase activity which is able to hydrolyse the sialic acid on the surface of the host cells,expose the biological recognition site of host cells,promote antigen presented,increase the production of cytokines and chemical factors surrounding tumor cells,and cause infiltration of lymphocytes,mononuclear cell,etc.To enhance the treatment effect of HN gene on melanoma,we also chose mouse telomerase reverse transcriptase promoter?mTERTp?and mouse tyrosinase promoter?mTyrp?to regulate the expression of HN gene.Telomerase reverse transcriptase promoter?TERT?is the most important component for the catalytic activity of telomerase.Changes of telomerase activity are closely related to aging and the occurrence of tumor cells.TERT often expresses in more than 90%tumors,most of the human and mouse cancer or tumor cell lines have shown a high expression of telomerase activity,so it is a broad spectrum marker of tumors.Tyrosinase?Tyr?is a key enzyme in melanoma formation,its expression and activity determines the speed and production of melanin,the high level of tyrosinase activity can lead to spots and the formation of melanoma.Further studies have found that Tyr promoter only expresses specifically in melanoma cells other than normal tissues.Therefore,promoters of TERT and Tyr possess the characteristics to regulate the downstream therapeutic gene HN.We designed a pair of PCR amplification primer and two pairs of RT-PCR amplification primer according to mTERT gene sequences?AF157502.1?,mTyr gene sequences?D00439.1?and NDV HN cDNA sequence?EF211815.1?downloaded from GenBank,and amplificated target genes from C57BL/6 mice thymus DNA,mouse melanoma B16 cells total RNA and pShuttle-CMV-HN plasmid respectively,then ligated the target fragments with the pMD-19T vector.After tested by double digestion and sequencing analysis,we named them pMD-mTERTp,pMD-mTyrp,pMD-HN.T4 DNA Ligase was used to link target fragments mTERTp,mTyrp,HN with pShuttle-IRES-hrGFP-2 respectively.Linearization shuttle vector with Pme I,and transfected it with pAdeasy-1 into BJ5183 cells,by homologous recombination,finally we obtained recombinant adenovirus plasmid.The recombinant adenovirus transfected HEK293 cells with lipidosome,and constructed a recombinant adenovirus with Hemagglutinin-neuraminidase?HN?gene which is co-regulated by mTERT and mTyr promoters named Ad-mTERTp-mTyrp-HN,after confirmed by RT-PCR and sequencing analysis,we collected the virus particles by CSCl density gradient ultracentrifugation and purification,and determined the recombinant adenovirus titers with TCID₅₀ method.The titers of Ad-HN,Ad-mTERTp-HN,Ad-mTyrp-HN,Ad-mTERTp-Tyrp-HN,Ad-GFP are 10^11.25TCID₅₀/mL?10¹2.52.5 TCID₅₀/mL?10^11.625TCID₅₀/mL?10^11.875 TCID₅₀/m L?10^12.125 TCID₅₀/m L respectively,which is enough for next step research.Through the Hochest33342/PI dying experiments have confirmed that the recombinant adenoviruses can infect of B16 cells.MTT assay had determined that the Ad-mTERTp-Tyrp-HN was able to inhibit the growth of B16 cells and the peak value occurs in the 4^th day,whose inhibition rate is significantly greater than the other control group recombinant adenoviruses.The results of western blotting have determined that HN protein expressed in B16 cells.The apoptosis rates of B16cells infected by recombinant adenoviruses Ad-HN,Ad-mTERTp-HN,Ad-mTyrp-HN,Ad-mTERTp-Tyrp-HN and control adenovirus Ad-GFP were28.03±0.45%;48.36±0.95%;39.12±0.78%;58.39±1.47%and 19.05±0.89%determined by flow cytometry.This is the first time that using two kinds of promoters to regulate HN gene to construct a new recombinant adenovirus vector system which is able to express exogenous gene efficiently and steadily.This research provided both theoretical and experimental basis for inducing tumor cells apoptosis in vitro by using recombinant adenovirus,and developed a broad spectrum vaccine for targeting therapy method for animal tumors.