Cloning Of Human Tumor Necrosis Factor-?(hTNF-?),Human Interferon-?(hIFN-?),Chicken Vasa Homolog(CVH) And Deleted In Azoospermia-like(DAZL),Construction Of Their Lentiviral Expression Vectors And In Vitro Expression Assay

Human tumor necrosis factor-?(hTNF-a)is a bioactive factor which has the strongest tumor-killing activity that ever found.However its application has been limited because of its evident side-effects,such as short half-life and less effectiveness at low doses and so on.Point mutated hTNF-a has high efficiency and low toxicity.As a type ? Interferon,human interferon-? has broad-spectrum antiviral activity,so it has been used to treat viral disease,tumour and autoimmune disease in clinic.Chicken primordial germ cells(PGCs),the precursor cells of gamete,could be used as efficient carrier to transfer foreign gene into genome or reproductive tract,since PGCs can targeting transfer into sex cell cord of heterozygote of embryo,then the hereditary character would be passed on to future generations.It is speculated that the combination of DAZL and/or CVH with a targeting gene to conduct into PGCs in vivo/in vitro may improve germline competence of the PGCs,which helps to overcome the problem of lower efficiency in production of transgenic chicken.Methods:(1)Peripheral venous blood was collected from healthy volunteers,lymphocyte was isolated and cultured,lipopolysaccharide(LPS)was added to culture medium to activates macrophages.Total RNA was extracted from the cultured lymphocyte by TRIzol regent,hTNF-?,hIFN-? gene was amplified by RT-PCR,and point mutation was made in hTNF gene.In addition,chicken CVH and DAZL gene were cloned from plasmid pMD18-T-CVH and pMD18-T-DAZL by PCR techniques.(2)By means of MultiSite Gateway techniques,lentiviral expression vectors of hTNF-? and hIFN?,CVH and DAZL were constructed,the first two vectors were regulated by ovalbumin promoter,while the latter two ones by EF1? promoter.(3)293FT cells were transfected by the above 4 lentiviral vectors,culture medium was collected to infect 293FT cells,and RT-PCR and western blot was used to detect the expression of hTNF-?,hIFN?,CVH and DAZL.The results showed that:(1)hTNF-a,hIFN-y,CVH and chicken DAZL gene were successfully cloned,and four entry vectors,or pENTRTM/D-TOPO-hTNF-?,pENTRTM/D-TOPO-hIFNy,pENTRTM/D-TOPO-CVH and pENTRTTM/D-TOPO-DAZL,were constructed.(2)Four lentiviral vectors of these 4 genes were aslo successfully constructed,which include pLenti6.4/OVP1548/V5/hTNF-a,pLenti6.4/OVP1548/V5/hIFNy,pLenti6.4/EF1?/V5/CVH and pLenti 6.4/EF1?/V5/DAZL.(3)RT-PCR and western blot assay verified that the expression of hTNF-?,hIFN-?,CVH and chicken DAZL in 293FT cells.This paper will lead to improve germline competence of transgenic chicken in which hTNF-?/hIFN-? gene is specifically expressed in ovduct.