Transformation And Amplification Of Human CD4 And HLA-DR0401 Expression Vectors And Their Expression In L929 Cells

1.Objectives and significanceRheumatoid arthritis(RA) is a chronic,multiple and general autoimmune disease,which is characterized by arthrosynovitis and symmetrically destructive arthropathy.The incidence of RA surround the globe is about 0.5%-1.0%and 0.4% in our country.Although the number of patients with RA is lower than that in foreign countries,it is estimated that the total number of patients with RA is as large as 60 million for the reason that the great population exists in our country.Furthermore, RA,to some extent is a kind of stubborn disease characterized by chronicity, advancement,and rodent.If untreated,in one or two years from the onset of the disorder,the patients would suffer bone destruction,disability and even death. Undoubtedly,the patients with RA will usually bear high mental stress,and the quality of life is also significantly lower than the normal ones.The definite etiology is not clear untill now time,but the key step of pathology about RA has been understanded.We are now grown to realize the onset of RA is closely related with hereditary factor other than environmental factor.With the development of immunogenetics,many studies have confirm that there is a definite correlation between RA and the human lymphocyte antigen(HLA).HLA complex locates at the short arm of the 6th chromatosome of human.HLA-Ⅱ,possessing a character of high polymorphism,is the leading element taking part in the specific recognition and immune response within human,including DR,DQ and DP that are found to have some correlation with the onset of RA at present.In the recent decades have witnessed the most significant advancement in the researches ofpathogenesis of RA is the revealing of HLA-DR4 which are found to have close relationship with the disease severity in RA and found to play 30%-50%role in the susceptivity of RA. Further researches on HLA-DR4 subtypes have shown that it is the subtypes such as DR4~*0401(that is DRβ1~*0401),0404,0405,0408 and DR1~*0101 have the chief hereditary influence in the onset of RA.These subtypes share the same epitope in the 70th to 74th ofβchain of DR1 and DR4(encoding by HVR).These especial amino acid sequence are QK/RRAA,so it is called "Shared Epitope".It may mediate the onset of RA by presenting antigen or reacting as antigen fragment.Recent years have shown that HLA-DQ3 too is important allele related with RA,especially HLA-DQ3~*0301(that is DQβ1~*0301)which is a marking gene of RA.Linkage disequilibrium exists between HLA-DQβ1~*0301 and most DR4 haplotype.Studies have shown that they have detected the important HLA-DR4-DQ3 haplotype in the RA population,which have five times higher levels of risk than the simple SE genes. Correlation analysis concerning HLA-DP and RA are relatively rare,some experts consider that apart from the important DR4,DP also could effect the susceptivity of the disease and could regulate the RA development.HLA-DP~*0401 is thought to the leading gene related with RA and DPβ1~*0201,0601 may be the risk factor in RA when the SE is negative.Comprehensive analysis have shown that RA is autoimmune disease correlated with multiple immunogenes.Although other genetic elements such as TCR gene,immunoglobulin gene,polypeptide transporting protein gene are related with RA,the role of HLA-Ⅱin the onset of RA is the most obvious. Since the pathogenesy of RA is unknown,now we are in the shortage of satisfactory,definitely effective and less toxic medication.In order to probe furtherly into the mechanisms such as etiopathogenisis,pathology,immunology and clinical mechanisms of RA which leading to founding the more of effective therapeutic regimens,favourable animal models are needed urgently.Now three animal models have been put into practice in the research of RA:one is collagen induced arthritis,the other is autoimmunity arthritis,and the last one is transplantation type arthritis.Collagen induced arthritis deserves the most frequent research.However,RA is the consequence of combination of genetic,infective, environmental,immunizing factors and RA animal models only put emphasis on one or several factors without one that could completely imitate the state of human RA. The selected RA animal models differ with human RA in the pathogenesis. Obviously,to choose an animal model that is the same or similar with human RA in the pathogenesis and pathology becomes increasingly important in the studies of RA.Transgenic animal is a kind of animal that the exogenous gene could be stably integrated into its chromosome genome by means of experiment inductions and transmitted to the next generation.When the exogenous gene is expressed in the body of the transgenic animal and the animal model whose phenotype is similar with the human diseases symptoms is cultivated,it can be called transgenic animal model, whose etiopathogenisis is clear(caused by the transgenic exogenous gene) and their symptom is single and similar to the patients.Several transgenic mouse models have been built in domestic and oversea.Therefore,the exploitation of transgenic mouse models become the hot spot in building RA animal models.Now the establishment of carring transgenic mouse related gene is at the stage of beginning and most laboratories still adopt the most classic way of micro-injection method.The construction of transgenic mouse animal model requires the consumption of a large sum of manpower and substances,so perfect preparations must be made during the structure and quality of the exogenous gene is one of the basic conditions the success of transgene.Exogenous gene used for transgene technologies must be self-express after transmitted into animals and can be identified through certain method beside their own genetic feature and clinical feature. Therefore,it is of primary importance in studying whether the exogenous gene could be expressed in cells in vitro.In the past twenty years,the experts,national and international,had begun to research the transfection of HLAⅡinto the mouse fibroblast and have achieved some achievements.The plasmids encoding HLA-DR0401 or human CD4 were transfected into the mouse fibroblast L929 respectively.HLA-DR0401 plays a major genetic role in the onset of RA.Moreover,CD4 is the co-receptor of MHCⅡ. Studies have shown that,the onset of RA and CD4~+T cells are closely related.CD4 antigen can combine with antigen polypeptides which are presented by antigen presenting cells.So CD4 antigen can elevate the ability ofuptaking HLA-DR antigen.The objective of the study was to transform and amplify human CD4 and HLA-DR0401 vectors,and to make them could be expressed in the mouse fibroblast L929,so that they could further pave the experimental foundation for the construction of RA transgenic mouse model.2.Methods and projects2.1 The transformation and amplification of pcDNA3-CD4 and pLNCX2-DR~*0401DH5αcompetent cells were kept in the iced bath,1μl plasmid was add into the suspension of competent cells,then mixed them and standed still in the iced bath for 30min,after 42℃aqueous bath for 90s,put them quickly in the iced bath for 2 min. In the system,added 500μl LB nutrient medium,37℃swing(150rpm) cultured 45 min.Put the transformed competent cells 100μl spreaded over the LB solid medium containing ampicillin.Then they were put into the fiat plate in the room temperature until the liquid completely be absorbed,upsided down the flat plate,cultivated in 37℃for 12-16h.After colonies were picked to be amplifed and cultured,the plasmids were extracted by following the instruction for the plasmid extraction kit.2.2 The identification ofpcDNA3-CD4 and pLNCX2-DR~*0401According to the construction map of plasmid,with the method of restriction enzyme first to make the plasmid was identified initially.The sequences ofpcDNA3-CD4 and pLNCX2-DR~*0401 were detected in Invitrogen company(Guangzhou) and TaKaRa company(Dalian) respectively.2.3 The cultivation and passage of L929 cell lineL929 cells were cultivated in the complete medium containing RPMI1640+10 %calf serum+1%double antibioticses.Before the passage,used trypsin-EDTA to digest exponential growth phase cells from the flask wall.Then added some fresh culture medium and adjusted the cell density.Then divided and went on cultivating them.Changed the culture medium every day since the process of passage was about two days.2.4 Transfection of L929 cells by LiposomeFollow the instruction on the Lipofectamine kit in the process of transfection and optimize the specific conditions.The details were as follows:digested and passaged the L929 cells in exponential phase of growth the day before transfection,usd 1mL /well inoculate in the six-well plate(cell density:4×10~5/mL).Mixed the pcDNA3-CD4 and pLNCX2-DR~*0401 respectively with liposome in the proportion of 1:3 when the cells grew to cover 90%of the surface.Incubated in the medium without serum for 20min and put them in the L929 cells for 6h.Then cultivated them in RPMI 1640 medium containing 20%fetal calf serum(FCS) for the next 48h.2.5 The expression and identification of humanCD4 and HLA-DR0401 in L929 cells.(1) Detection by flow cytometry1×10~6 transfected cells were collected and washed once with cold PBS in 4℃, suspended cells with 100μl PBS,added respectively 20μl FITC marked anti-human CD4 monoclonal antibody(RPA-T4) and PE marked anti-human HLA-DR monoclonal antibody(LN3),cultivated them 1h away from the light in 4℃,washed twice using PBS,and then suspended cells with 200μl PBS for detection.(2) Detection by direct immunofluorescenceFilm preparation of the transfected cells.Fixed in 4%paraformaldehyde 30min, washed three times with PBS,dried it and then added 20μl FITC marked anti-human CD4 monoclonal antibody(RPA-T4) and PE marked anti-human HLA-DR monoclonal antibody(LN3) respectively,cultivated them away from the light in 4℃over night,washed with PBS three times and mounted them in 50%glycerine.Put them under fluorescence microscope to be observed.3.Results and analysis3.1 The concentration and purity of the amplified plasmidAfter transformation and amplification,the extractive of pcDNA3-CD4 plasmid with its A260/A280=1.86,concentration:474ng/μl,and pLNCX2-DR~*0401 plasmid with its A260/A280=1.90,concentration:182ng/μl.The results showed that the plasmids which had a better quality and a higher purity were acquired after transformation.3.2 The identification ofpcDNA3-CD4 and pLNCX2-DR~*0401(1) The identification result of pcDNA3-CD4 by restriction endonuclease digestion and DNA sequencingPlasmid pcDNA3-CD4 produced a segment as long as about 8.5kb after it was cut by single enzyme,according to the construction map ofplasmid,pcDNA3-CD4 was cut by double enzymes with NruⅠand PinAⅠ,it could result in two segments, 2250bp and 6221bp respectively.The identification result by restriction endonuclease digestion was correct.The sequence analysis of plasmid pcDNA3-CD4 and compared with CD4 gene(NM-000616 ) sequence logging by GenBank proved that the DNA sequencing was completely correct.(2) The identification result of pLNCX2-DR~*0401 by restriction endonuclease digestion and DNA sequencingPlasmid pLNCX2-DR~*0401 produced a segment as long as about 8.3kb after it was cut by single enzyme.According to the construction map ofplasmid,pLNCX2-DR ~*0401 was cut by double enzymes with BglⅡand NotⅠ,it could result in two segments,2233bp and 6102bp respectively.The identification result by restriction endonuclease digestion was correct.The sequence analysis of plasmid pLNCX2-DR ~*0401 and compared with HLA-DRαgene(NM-019111) sequence logging by GenBank proved that the sequence detection was completely correct.The detection of HLA-DRβ1 sequence by a reversed design came to an end because the advanced structure existed in the sample.3.3 The cultivation and the passage of L929 cell lineThe growth condition of L929 cells proved to be fine with their shapes varied like fusiform,polygon or flat aster。3.4 Transfection of L929 cells by Lipofectamine 2000After the transfection for 48h,about 5%of the cells died,the shapes of the cells did not significantly transform compared with the condition before transfection.3.5 The detection of human CD4 transfected cells by flow cytometryWe detected the expression of human CD4 protein in transfected L929 cells by FITC marked anti-human CD4 monoclonal antibody(RPA-T4),and make a comparison with L929 cells transfected pcDNA3-CD4 plasmid without adding antibody or the non-transfected L929 cells with antibody as controls.The results showed that we did not detect the fluorescence labeling positive cells in control groups,but within the transfected L929 cells with human CD4 plasmid,there was a percent of 61 marked positive.3.6 The detection by flow cytometry in HLA-DR0401 transfected cellsWe detect the expression of human HLA-DR0401 protein in transfected L929 cells by PE marked anti-human HLA-DR monoclonal antibody(LN3) and make a comparison with L929 cells transfected pLNCX2-DR~*0401 plasmid without adding antibody or the non-transfected L929cells with antibody as controls.The results showed that we did not detect the fluoresce-ence labeling positive cells in control groups,but within the transfected L929 cells with HLA-DR0401 plasmid,there was only a percent of 1 marked positive.3.7 The detetion of the expression of human CD4 and HLA-DR by direct immunofluorescenceIt was detected that the expression of human CD4 protein in transfected L929 cells through the use of direct immunofluorescence by FITC marked anti-human CD4 monoclonal antibody(RPA-T4),and we could make a comparison with L929 cells transfected pcDNA3-CD4 plasmid without adding antibody or the non-transfected L929 cells with antibody as controls.The results showed that we did not detect the fluorescence labeling positive cells in control groups,but obvious green fluorescence could be detected within the transfected human CD4 plasmid in L929 cells.This proves that the human CD4 protein could be expressed in cells.It was not detected that the fluorescent labelling in L929 cells transfected with HLA-DR plasmid through the use of direct immunofluorescence by PE marked anti-human HLA-DR monoclonal antibody(LN3).4.ConclusionThe transformation and amplification of expression carrier of human CD4 and HLA-DR0401 was successful,but the transfection of the mouse fibroblast cells L929 was not perfect.The plasmid encoding human CD4 was expressed with highefficiency, it can react as exogenous gene of transgenic experiment.However,the plasmid encoding HLA-DR0401 had low transfection efficiency,it must be refining. The research laid the experimental foundation for further construction of transgenic mouse model.