本文選題：T-bet　切入點：單克隆抗體　出處：《江蘇大學》2007年碩士論文

【摘要】： 人類轉錄因子T-bet(T-box expressed in T cells，T-bet)基因，作為Th1細胞特異性的轉錄因子，在Th1細胞分化中誘導IFN-γ分泌，上調IL-12Rβ2的表達，同時抑制IL-4的表達。此基因的開放閱讀框架包含1607bp，可編碼530個氨基酸殘基，其中有一個由189個氨基酸組成的能與T盒DNA結合的結構域，主要在肺、脾臟及胸腺表達，其分布說明T-bet的表達具有限制性。 目的 克隆并鑒定人T-bet基因；構建原核表達載體并在體外表達T-bet蛋白，純化蛋白作為抗原免疫小鼠，制備并初步鑒定單克隆抗體；構建攜帶穿膜序列(Tat)和目的基因(T-bet)的表達載體，通過大腸埃希菌表達TAT-T-bet蛋白，將此重組蛋白轉染THP-1細胞，初步觀察T-bet在調節(jié)Th1／Th2平衡中的作用。 方法 (1)從人外周血分離單個核細胞，抽提總RNA，逆轉錄cDNA，PCR擴增獲得人T-bet基因；將T-bet基因克隆至pGEM-T載體，進行單、雙酶切鑒定及序列分析。 (2)將T-bet基因克隆到pQE30原核表達載體，在大腸桿菌M15中表達，獲得帶有His標簽的62KD左右的T-bet融合蛋白；利用Bio-Rad公司的IMAC柱純化T-bet蛋白；以T-bet蛋白作為抗原免疫Balb／c小鼠，取免疫小鼠的脾細胞與SP2／0細胞融合，在HAT培養(yǎng)基中培養(yǎng)并篩選得到分泌抗T-bet單克隆抗體的雜交瘤細胞株。 (3)T-bet基因克隆到pIRES_2-eGFP真核表達載體，將重組質粒pT-bet通過電穿孔法導入人單核巨噬細胞株THP-1，并以空質粒電轉THP-1細胞作為陰性對照；用G418篩選電轉陽性細胞，得到單個克隆陽性細胞株。 (4)將穿膜序列TAT重組到pQE30載體上，構建具有穿膜功能的原核表達載體；再將T-bet基因重組到pQE30-TAT載體上，在大腸埃希菌M15中表達具有穿膜功能的T-bet蛋白；將純化的穿膜蛋白加入THP-1細胞培養(yǎng)體系，觀察穿膜現象及效率；將蛋白轉染的THP-1細胞與外周血CD4~+T細胞共培養(yǎng)，初步觀察TAT-T-bet對THP-1細胞的作用，探討轉染的THP-1細胞對CD4~+T細胞分化的影響。 結果 (1)成功克隆了包括1607bp編碼區(qū)的人T-bet基因全長，并在大腸埃希菌有效表達，獲得了純化的T-bet蛋白。 (2)成功制備了人T-bet單克隆抗體，經三個月克隆和篩選，獲得三株穩(wěn)定分泌抗體的雜交瘤細胞株，經ELISA和Western-blot鑒定具有良好的反應性及特異性。 (3)構建具有穿膜功能的pQE30原核表達載體，獲得T-bet穿膜蛋白，并以濃度和時間依賴的方式轉染THP-1細胞，轉染的THP-1細胞與CD4~+T細胞共培養(yǎng)，能誘導CD4~+T細胞分泌IFN-γ，促進Th1細胞的分化。 結論 獲得了人T-bet克隆并在大腸埃希菌中有效表達。制備和純化了人T-bet蛋白，并以此為抗原，應用雜交瘤技術制備了分泌T-bet抗體的雜交瘤細胞株。應用蛋白轉染技術，成功將重組人T-bet蛋白轉染THP-1細胞株，籍此考察和分析了轉有T-bet蛋白的THP-1細胞對Th1細胞的調節(jié)作用，且表明T-bet蛋白轉染的THP-1可通過上調IFN-γ的表達而促進Th1活性。
[Abstract]:Human transcription factor T - bet ( T - box expressed in T cells , T - bet ) gene , which is a transcription factor specific to Th1 cell , induces IFN - 緯 secretion in Th1 cell differentiation , up - regulation of IL - 1212尾2 , and inhibits IL - 4 expression . The open reading frame of this gene contains 1607bp , which can encode 530 amino acid residues , and one of 189 amino acids can be expressed in the lung , spleen and thymus , and its distribution indicates that the expression of T - bet is restricted .






Purpose






cloning and identifying human T - bet gene ;
constructing a prokaryotic expression vector and expressing the T - bet protein in vitro , purifying the protein as an antigen immune mouse , preparing and preliminarily identifying the monoclonal antibody ;
The recombinant protein was transfected into THP - 1 cells by expression of TAT - T - bet protein by E . coli , and the role of T - bet in regulating Th1 / Th2 balance was preliminarily observed .






method






( 1 ) separating mononuclear cells from human peripheral blood , extracting total RNA , reverse transcription cDNA and PCR amplification to obtain human T - bet gene ;
The T - bet gene was cloned into the T - T vector , and the single , double enzyme digestion and sequence analysis were carried out .






( 2 ) cloning the T - bet gene into a prokaryotic expression vector of pQE30 , and expressing in Escherichia coli M15 to obtain a T - bet fusion protein with a His tag of about 62KD ;
Purification of T - bet protein using IMAC column of Bio - Rad Company ;
Balb / c mice were immunized with T - bet protein as antigen . The spleen cells of immunized mice were fused with SP2 / 0 cells . The hybridoma cell lines secreting anti - T - bet monoclonal antibody were cultured and screened in the medium .






( 3 ) cloning the T - bet gene into a pIRES _ 2 - eGFP eukaryotic expression vector , introducing the recombinant plasmid pT - bet into the human mononuclear phagocyte THP - 1 through electroporation method , and carrying out electric transformation THP - 1 cell with an empty plasmid as a negative control ;
The positive cells were screened by G418 , and a single clone positive cell line was obtained .






( 4 ) the membrane - penetrating sequence TAT is recombined into a pQE30 carrier , and a prokaryotic expression vector with a membrane penetrating function is constructed ;
then the T - bet gene is recombined on the pQE30 - TAT vector , and the T - bet protein with the membrane penetrating function is expressed in the Escherichia coli M15 ;
adding purified membrane protein into THP - 1 cell culture system to observe the phenomenon and efficiency of membrane penetration ;
The effect of TAT - T - bet on THP - 1 cells was investigated by co - culture of THP - 1 cells transfected with protein and CD4 ~ + T cells in peripheral blood . The effects of transfected THP - 1 cells on the differentiation of CD4 ~ + T cells were investigated .






Results






( 1 ) The full length of human T - bet gene including 1607bp coding region was cloned successfully , and the purified T - bet protein was obtained in Escherichia coli .






( 2 ) The monoclonal antibody of human T - bet was successfully prepared , and three hybridoma cell lines stably secreting antibodies were obtained by three - month cloning and screening .






( 3 ) constructing prokaryotic expression vector of pQE30 with membrane penetrating function , obtaining T - bet membrane protein , and co - culturing THP - 1 cells in concentration and time - dependent manner , co - culturing THP - 1 cells transfected with CD4 + T cells , and inducing CD4 + T cells to secrete IFN - gamma to promote differentiation of Th1 cells .






Conclusion






A hybridoma cell line secreting T - bet antibody was prepared and purified by using hybridoma technique . THP - 1 cell line transfected with T - bet protein was successfully transfected into THP - 1 cell line . THP - 1 transfected with T - bet protein was successfully transfected into THP - 1 cell line .

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346

【引證文獻】

相關碩士學位論文 前1條

1 劉小娟;參芪扶正注射液對再生障礙性貧血小鼠T-bet及IEN-γ表達的影響[D];遼寧醫(yī)學院;2012年

，

本文編號：1714981