人T-bet基因克隆及其功能的初步研究

发布时间：2018-04-05 13:35

本文选题：T-bet　切入点：单克隆抗体　出处：《江苏大学》2007年硕士论文

【摘要】： 人类转录因子T-bet(T-box expressed in T cells，T-bet)基因，作为Th1细胞特异性的转录因子，在Th1细胞分化中诱导IFN-γ分泌，上调IL-12Rβ2的表达，同时抑制IL-4的表达。此基因的开放阅读框架包含1607bp，可编码530个氨基酸残基，其中有一个由189个氨基酸组成的能与T盒DNA结合的结构域，主要在肺、脾脏及胸腺表达，其分布说明T-bet的表达具有限制性。目的克隆并鉴定人T-bet基因；构建原核表达载体并在体外表达T-bet蛋白，纯化蛋白作为抗原免疫小鼠，制备并初步鉴定单克隆抗体；构建携带穿膜序列(Tat)和目的基因(T-bet)的表达载体，通过大肠埃希菌表达TAT-T-bet蛋白，将此重组蛋白转染THP-1细胞，初步观察T-bet在调节Th1／Th2平衡中的作用。方法 (1)从人外周血分离单个核细胞，抽提总RNA，逆转录cDNA，PCR扩增获得人T-bet基因；将T-bet基因克隆至pGEM-T载体，进行单、双酶切鉴定及序列分析。 (2)将T-bet基因克隆到pQE30原核表达载体，在大肠杆菌M15中表达，获得带有His标签的62KD左右的T-bet融合蛋白；利用Bio-Rad公司的IMAC柱纯化T-bet蛋白；以T-bet蛋白作为抗原免疫Balb／c小鼠，取免疫小鼠的脾细胞与SP2／0细胞融合，在HAT培养基中培养并筛选得到分泌抗T-bet单克隆抗体的杂交瘤细胞株。 (3)T-bet基因克隆到pIRES_2-eGFP真核表达载体，将重组质粒pT-bet通过电穿孔法导入人单核巨噬细胞株THP-1，并以空质粒电转THP-1细胞作为阴性对照；用G418筛选电转阳性细胞，得到单个克隆阳性细胞株。 (4)将穿膜序列TAT重组到pQE30载体上，构建具有穿膜功能的原核表达载体；再将T-bet基因重组到pQE30-TAT载体上，在大肠埃希菌M15中表达具有穿膜功能的T-bet蛋白；将纯化的穿膜蛋白加入THP-1细胞培养体系，观察穿膜现象及效率；将蛋白转染的THP-1细胞与外周血CD4~+T细胞共培养，初步观察TAT-T-bet对THP-1细胞的作用，探讨转染的THP-1细胞对CD4~+T细胞分化的影响。结果 (1)成功克隆了包括1607bp编码区的人T-bet基因全长，并在大肠埃希菌有效表达，获得了纯化的T-bet蛋白。 (2)成功制备了人T-bet单克隆抗体，经三个月克隆和筛选，获得三株稳定分泌抗体的杂交瘤细胞株，经ELISA和Western-blot鉴定具有良好的反应性及特异性。 (3)构建具有穿膜功能的pQE30原核表达载体，获得T-bet穿膜蛋白，并以浓度和时间依赖的方式转染THP-1细胞，转染的THP-1细胞与CD4~+T细胞共培养，能诱导CD4~+T细胞分泌IFN-γ，促进Th1细胞的分化。结论获得了人T-bet克隆并在大肠埃希菌中有效表达。制备和纯化了人T-bet蛋白，并以此为抗原，应用杂交瘤技术制备了分泌T-bet抗体的杂交瘤细胞株。应用蛋白转染技术，成功将重组人T-bet蛋白转染THP-1细胞株，籍此考察和分析了转有T-bet蛋白的THP-1细胞对Th1细胞的调节作用，且表明T-bet蛋白转染的THP-1可通过上调IFN-γ的表达而促进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 - 纬 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 .

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

【引证文献】