【作者】 黄河；

【导师】 谭岩；

【作者基本信息】 吉林大学 ， 免疫学， 2006， 博士

【摘要】 早期造血生长因子FL是酪氨酸激酶Ⅲ受体(Flt3)的配体,与受体结合后能够调节最原始的造血干/祖细胞的增殖和分化,动员和刺激髓系及淋巴祖细胞、DCs和NK细胞增殖和分化,是树突状细胞(DC)最为重要的生长因子;可与多种细胞因子协同,提高造血功能,与IL-2、IL-4、IL-12、IFN-γ等抗肿瘤因子有协同效应,从而在机体抵抗肿瘤的免疫过程中起重要作用。本文从K562细胞中分离出人FL的胞外段基因(hsFL),与pcDNA3表达载体相连,构建了pcDNA3/hsFL真核表达质粒,脂质体转染Hela细胞后进行RT-PCR、Western blot、ELISA及一系列生物学活性鉴定的检测;同时还将hsFL与原核表达载体pQE30相连构建了原核表达质粒,并进行了表达和纯化。结果表明:本实验获得的hsFL cDNA序列与Genbank的登录结果一致,有两处点突变,但所表达的氨基酸未变;发现了两种hsFL的剪变体,对这两种剪变体的研究目前正在进行中。pcDNA3/hsFL真核表达质粒转染Hela细胞后,经RT-PCR验证细胞内有目的基因的mRNA存在,说明能够有效地转录hsFL基因;Western blot有特异条带出现,与预期位置一致;ELISA法测得72h后培养上清中的hsFL含量为56.8ng/ml/1×106cells,并且分泌的hFL能促HL-60细胞增殖,在无血清培养的条件下还能促进Raji细胞的生存和抑制细胞凋亡的发生。说明通过该真核表达系统表达出的蛋白质具有良好的生物学活性。构建了原核表达质粒pQE30/hsFL,表达出可溶型FL蛋白,并进行了纯化。原核表达蛋白Western blot在预期位置出现条带,造血集落形成实验证实原核系统表达的hsFL蛋白与SCF、GM-CSF、IL-3有协同作用,表现出良好的生物学活性。更多还原

【Abstract】 Hemopoietic growth factor Flt3 ligand(FL)is a type Ⅰtransmembrane protein.Thenative human FL is a 65KD nondisulphide-linked homodimeric glycoprotein comprisedof 30KD subunits , each subunit containing 12KD of N-linked and O-linked sugars.Thehuman FL gene encodes a 235-amino acid consisting of four domains:1) an N-termial26-residue signal peptide,2) a 156-residue extracellular domain,3)a 23-amino acidtransmembrane domain,and 4) a 30-residue cytoplasmic domain.FL is similar in structureto the haematopoietic growth factors M-CSF and c-kit ligand,they all belong to the shortchain helical bundle structural subfamily,and there is only one receptor binding site perligand subunit,but differs from them in not being species-specific with regard to itsbinding.Its was found to exhibit 72% homology with mouse FL at the amino acid level.The coding region of human FL encompasses 8 exons.It was certificated by genemutation test that 1-5 exons contains all necessary information to bring into full play inbiologic function.In the mRNA of an alternately spliced sixth exon,that introduced a stopcodon at the end of the extracellular domain ,thereby generating a soluble yetbiologically active form of FL.Transmembrane protein is coded by mRNA that hasn’t thesixth exon,playing the role on the cell membrane,which can be cleaved proteolytically toyield a soluble form by protease site.Multiple splice-variant forms of hFL mRNA havebeen identified for alternately spliced,thereby may be generating many kind of proteinisoform.hsFL protein is a soluble form that be coded by extracellular domain gene.FL is the most important growth factor for dendritic cells,it affects the growth anddifferentiation of pluripotent haematopoietic stem and progenitor cells,as well as anumber of lineages in the lymphoid, myeloid,NK and dendritic cells by binding Flt3receptor.A synergistic effect with a wide range of cytokine,to promote hematopoieticcapacity .Likewise,FL synergizes with anti-tumor factor such as IL-2、IL-4、IL-12、IFN-γ,to play a critical role in the anti-tumour immunity.The purpose of the research is focused on constructing of the eukaryotic expressionplasmid pcDNA3/hsFL and prokaryotic expression plasmid pQE30/hsFL,detecting theexpression of recombinant and the biological activity of hsFL protein..The construction of eukaryotic expression plasmid pcDNA3/hsFLThe total RNA was extracted from K562 cells by Trizol, and hsFL cDNA wascloned by RT-nested PCR , the PCR products were subcloned into pGEM-T forsequencing Target sequence was inserted into pcDNA3 to construct pcDNA3/hFL.Screening the positive clone by identification of restriction enzyme digestion and PCR.The results showed that hsFL cDNA sequence in our study was in accordance withthe report of Genbank.There were two pointmutation in sequence,but no change of theamino acid after translation. After evaluated by sequencing,target sequence was insertedinto pcDNA3 to construct eukaryotic recombined expressing plasmid pcDNA3/ hFL.In our study,we found two new splice-variant forms of hFL mRNA.There were the“b”product of 519bp and “c”product of 435bp.The “b”produc was deleted a C in thesecond exon and 85bp in the fifth exon,inserted 28bp intron sequence befor the thirdexon.The “c”product was inserted 28bp intron sequence like the “b”product and deletedthe full fifth exon.Although the third and fourth exon are intac,it is probable that thebiological activity may be changed for all or part deletion of the important fifth exon anddisplacement of ORF.We are keeping the investigation about protein expression andbiological activity of the two splice-variant forms, the relationship between proteinfunction and structure.Expression and function research of pcDNA3/hsFLHela cells were transfected with the recombinant by Lipofectamine2000. Collectedthe supernatant and cells after 72h of transfection.The transcription of hFL in thetransfected Hela cells was assayed by RT-PCR. The existence and size of hsFL proteinwere assessed in SDS-PAGE and silver staining,and the immunogenicity was detected byWestern blot.The content of hFL protein in the culture supernatant was assayed byELISA, the biological activity of hFL protein was assayed by proliferation experimentand survival promoting experiment.Hela cells 8×104 per well were put in 24-well plates.The optimal dose of liposomesis 1ul per well.The cells after transfected growth good and there were a little of cells died.The transcription of hsFL in the transfected Hela cells was assayed by RT-PCR usinginside primer2. The result showed that had a special band in the anticipated position witha great quantity. It was indicated that the hsFL gene was transcripted effectively intransfected Hela cells.After SDS-PAGE and silver staining,we found a 30KD proteinband in accordance with the anticipated position and there was a special band in identicalsite by Western blot assay.Above research data demonstrated that transfected Hela cellssecreted hsFL into supernatant and the protein had immunogenicity. The content of hFLprotein in supernatant was 56.8ng/ml/1×106cells in 72h after transfected. by ELISA.Using the characteristics that Raji and HL-60 cells express high level Flt3 receptoron cell surface,we acted the two cell lines as object to study the biological activity:different amount of supernatant(50ng/ml,200ng/ml)were respectively added into 104 cellculteres(IMDM,free serum) of Raji cell lines.The cultivation was maintained for 5 daysto count the vital cells and to assess apoptosis by FCM.The supernatant(0-100ng/ml)wererespectively added into 5 × 103cell culteres(IMDM,10%FCS)of HL-60 cells. Thecultivation was maintained for 68h and 72h respectively added into MTT andDMSO,detected the OD595 valuesThe results showed that the Raji cells in control group were all dead after 72h and inhsFL group had a part of cells survival after 5days.The apoptosis rate was decreased inhsFL group (p<0.01) .hsFL displayed an antiapoptotic activity. MTT assay dataindicated that hsFL (10-100ng/ml )promoted the growth of HL-60 cells(p<0.05).The construction,expression and function research of prokaryoticexpressing plasmid pQE30/hsFLAs the important growth factor of DCs and hemopoietic cells,FL play a key role inanti-tumor Immunity and cultivation of LTC-IC.It is difficult to obtain a great quantityof FL from animal or human becase its level is very low in vivo,so using extracorporealexpression system is necessary.We carried out PCR by intra-primer 3 and pcDNA3/hsFL as template to clone thehsFL gene, target sequence was inserted into pQE30 to construct prokaryotic recombinedexpressing plasmid pQE30/hsFL.The plasmid was transformated into E.coli M15 andscreened the high expressing recombinant by SDS-PAGE. Western blot, solubitwlityanalysis, to separate and wash inclusion bodies were completed. Purification of hsFLprotein by nickel affinity chromatography,determinations the content of pure protein byBCA assay, assessment of biological function by colony forming test.The results showed that 18KD hsFL protein was expressed in M15 with inclusionbody form. There was a special band in identical site by Western blot assay.The contentof hsFL was 400ug/ml.It is made clear that hsFL gene was expressed with highperformance in the prokaryotic expressing system.We separated PBMC from CB,added different combination of SCF,GM-CSF,IL-3,hsFL into methylcellulose of PBMC.The effect of hsFL on pluripotent haematopoieticstem and progenitor cells was observed .The tested data (mean±standard deviation)were analyzed by t test.Compared to the control,the number of CFU was increasedobviously ( p<0.05or p≤0.01) in cytokine groups;the proliferation effect of hsFL+GM-CSF+IL-3+SCF group was the strongest.The experiments showed that hsFLhad asynergistic effect with SCF,GM-CSF,IL-3 .Conclusion:We cloned hsFL gene from K562 cells , constructde the eukaryoticexpression plasmid pcDNA3/hsFL and prokaryotic expression plasmid pQE30/hsFL,detected the expression of recombinant and the biological activity of hsFL protein..hsFLgene in the two expressing system was transcripted and translated effectively, the hsFLprotein showed obvious biological activity and immunogenicity.It is valuble to keep theresearch about the characteristics of the two new splice-variant forms.Innovation point:1)hsFL gene was cloned from K562 lines by RT-nested PCR;2)Twosplice-variant forms were found firstly,the deep study of them may be valuble tocomprehend the mechanisms of FL expression and adjustment.更多还原