人HLX基因克隆表达及其功能的初步研究

发布时间：2018-04-27 23:12

本文选题：HLX + 原核表达　；参考：《江苏大学》2009年硕士论文

【摘要】： 目的: 克隆人HLX(H2.0-like homeobox gene,HLX)基因,原核表达并纯化HLX蛋白;制备针对人HLX融合蛋白的兔源性多克隆抗体并加以鉴定;定量PCR检测胃癌病人外周血单个核细胞中HLX mRNA的表达水平,以探讨HLX基因表达水平对机体Th1/Th2平衡状态的影响及其与胃癌发生发展的关系。方法: (1)采用RT-PCR方法从人脐血单个核细胞获得HLX基因,克隆至pMD19-T载体,转化E.coli DH5α宿主菌,挑取菌落进行酶切鉴定,将初步确定的阳性克隆进一步测序确认。 (2)以PCR的方法从HLX阳性克隆中获得HLX基因的ORF区全长,连接至pQE30原核表达载体,转化E.coli DH5α宿主菌,挑取菌落进行酶切和测序鉴定。 (3)将阳性重组质粒pQE30-HLX转化入E.coli M15菌中,经异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达,所表达的HLX融合蛋白经SDS-PAGE电泳及Western blot鉴定。大量诱导表达HLX融合蛋白,利用Profinity IMAC Ni-Charged Resin亲和层析柱分离纯化,以HLX蛋白作为抗原免疫家兔,收集含抗HLX多克隆抗体的兔血清,并用ELISA和Westernblot法鉴定抗体的效价和特异性。 (4)定量PCR检测胃癌患者外周血单个核细胞(peripheral blood mononuclearcells,PBMC)中HLX的表达,并与健康对照组进行比较研究,同时分析胃癌患者HLX的表达与T-bet、GATA-3表达的关系,以从转录因子水平了解可能存在于胃癌患者机体的Th1/Th2平衡失调状态。结果: (1)成功克隆了人HLX基因,构建了pMD19-T-HLX质粒。 (2)成功克隆人HLX基因ORF区全长,构建pQE30-HLX原核表达质粒。测序结果显示,连接至pQE30载体的目的片段为HLX的ORF区,全长1467bp,编码488个氨基酸残基,与GenBank中发表的序列完全一致。经诱导和纯化获得HLX融合蛋白,大小约为55kD。 (3)成功制备了针对人HLX蛋白的多克隆抗体,经ELISA和Western blot鉴定,具有良好的反应性及特异性,也表明HLX融合蛋白具有良好的免疫原性。 (4)实时荧光定量PCR检测了胃癌患者PBMC的HLX表达水平,较之健康对照组,其表达量有所下降。同法检测了胃癌患者T-bet、GATA-3表达的情况,分析了与两种基因间的相关性。结论: 成功构建了pQE30-HLX原核表达质粒并在大肠埃希菌中有效表达;获得人HLX融合蛋白,并以此为抗原,制备了特异性多克隆抗体;检测了HLX在胃癌患者PBMC的表达水平,较之健康对照组有所下降,并其表达水平与其他Th1型细胞因子或转录因子T-bet呈正相关,与GATA-3呈负相关,推测其可能与胃癌的发生发展存在一定的关系。
[Abstract]:Objective: Cloning of human HLX(H2.0-like homeobox gene, prokaryotic expression and purification of HLX protein, preparation and identification of rabbit polyclonal antibody against human HLX fusion protein, quantitative PCR detection of HLX mRNA expression in peripheral blood mononuclear cells of gastric cancer patients, To investigate the effect of HLX gene expression level on the balance of Th1/Th2 and its relationship with the occurrence and development of gastric cancer. Methods: 1) HLX gene was obtained from human umbilical cord blood mononuclear cells by RT-PCR method, cloned into pMD19-T vector, transformed into E.coli DH5 伪 host bacteria, and identified by enzyme digestion. The positive clones were further sequenced. (2) the whole ORF region of HLX gene was obtained from HLX positive clone by PCR, ligated to the prokaryotic expression vector of pQE30, transformed into E.coli DH5 伪 host strain, digested by enzyme and sequenced. The positive recombinant plasmid pQE30-HLX was transformed into E.coli M15 strain and was induced to express by isopropyl 尾 -D-thiogalactopyranoside pQE30-HLX. The expressed HLX fusion protein was identified by SDS-PAGE electrophoresis and Western blot. A large number of HLX fusion proteins were induced and purified by Profinity IMAC Ni-Charged Resin affinity chromatography. Rabbits were immunized with HLX protein as antigen. Rabbit sera containing polyclonal antibodies against HLX were collected. The titers and specificity of the antibodies were identified by ELISA and Westernblot methods. The expression of HLX in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer was detected by quantitative PCR and compared with that of healthy controls. The relationship between the expression of HLX and the expression of T-beta-GATA-3 in gastric cancer patients was also analyzed. In order to understand the imbalance of Th1/Th2 in patients with gastric cancer from the transcriptional factor level. Results: The human HLX gene was cloned successfully and the pMD19-T-HLX plasmid was constructed. The ORF region of human HLX gene was cloned successfully and the prokaryotic expression plasmid of pQE30-HLX was constructed. The result of sequencing showed that the target fragment ligated to the pQE30 vector was the ORF region of HLX, with a length of 1467bp, encoding 488 amino acid residues, which was identical to the sequence published in GenBank. HLX fusion protein was obtained by induction and purification, and the size of the fusion protein was about 55 KD. The polyclonal antibody against human HLX protein was successfully prepared, which was identified by ELISA and Western blot and showed good reactivity and specificity. It also showed that HLX fusion protein had good immunogenicity. The expression of PBMC HLX in patients with gastric cancer was detected by real-time fluorescence quantitative PCR, which was lower than that in healthy controls. The expression of T-bett GATA-3 was detected by the same method in patients with gastric cancer, and the correlation between the two genes was analyzed. Conclusion: The prokaryotic expression plasmid of pQE30-HLX was successfully constructed and effectively expressed in Escherichia coli. The fusion protein of human HLX was obtained and used as antigen to prepare specific polyclonal antibody. The expression level of HLX in PBMC of gastric cancer patients was detected. Compared with the healthy control group, the expression level was positively correlated with other Th1 type cytokines or transcription factor T-bet, and negatively correlated with GATA-3. It may be related to the occurrence and development of gastric cancer.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.11

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