小鼠趋化因子MIP-1α和共刺激分子B7-1基因重组慢病毒载体的构建及鉴定

发布时间：2019-01-12 15:03

【摘要】：【目的】肿瘤基因治疗的关键是载体的选择,慢病毒载体是以人类免疫缺陷病毒(HIV)为基础发展起来的基因治疗载体,它对分裂期细胞和非分裂期细胞均具有感染能力,能够整合到宿主细胞的基因组中,可以在体内长期稳定表达,还具有容纳外源性目的基因片段大、免疫反应小等优点。本研究采用慢病毒作为载体,构建小鼠趋化因子MIP-1α和共刺激分子B7-1基因重组慢病毒载体,鉴定其在293T细胞中的表达,为双基因治疗淋巴瘤的实验研究奠定基础。【方法】 1.第一部分,自行设计引物合成小鼠MIP-1α和B7-1基因的全长编码序列cDNA,采用巢式PCR扩增目的基因,使用Age I酶对慢病毒载体进行酶切消化,将目的基因与经酶切线性化的慢病毒载体进行定向连接,其产物转化大肠杆菌DH5α感受态细胞,对长出的阳性克隆进行PCR鉴定和直接测序序列分析,测序结果与目标基因序列完全一致的即为构建成功的目的基因质粒。然后将构建好的目的基因质粒进行超纯去内毒素抽提。 2.第二部分,MIP-1α和B7-1目的基因质粒分别转染293T细胞,48小时后,观察荧光报告基因的绿色荧光蛋白(GFP)表达,并采用Western Blot法检测目的基因质粒在293T细胞中的表达。 3.第三部分,采用pGC-LV重组慢病毒载体和包装质粒pHelper 1.0载体、pHelper 2.0载体组成的三质粒系统,共转染293T细胞,培养48 h后,收集富含慢病毒颗粒的细胞上清液,对其浓缩和纯化得到高滴度的慢病毒浓缩液。取10μl慢病毒浓缩液转染293T细胞,提取总RNA,逆转录获得cDNA,应用实时荧光定量PCR检测慢病毒浓缩液的滴度。【结果】 1.成功构建小鼠趋化因子MIP-1α和共刺激分子B7-1基因重组慢病毒载体,目的基因测序结果与目标序列完全一致。 2. MIP-1α和B7-1目的基因质粒转染293T细胞后,均可观察到荧光报告基因的绿色荧光蛋白表达,Western Blot法成功检测到MIP-1α、B7-1目的基因质粒在293T细胞中表达。 3.三质粒系统成功共转染293T细胞,包装出高滴度的MIP-1α、B7-1基因重组慢病毒浓缩液,实时荧光定量PCR证实MIP-1α、B7-1基因重组慢病毒载体的滴度均达2.00E+8 TU/ml。【结论】本研究成功构建并包装出高滴度的小鼠趋化因子MIP-1α和共刺激分子B7-1基因重组慢病毒载体,为淋巴瘤双基因治疗的实验研究奠定了基础。
[Abstract]:[objective] the key of tumor gene therapy is the selection of vector. Lentivirus vector is a gene therapy vector developed on the basis of human immunodeficiency virus (HIV). It has the ability to infect both mitotic and non-mitotic cells. It can be integrated into the genome of host cells and can be expressed stably in vivo for a long time. It also has the advantages of containing large foreign target gene fragments and small immune response. In this study, the recombinant lentivirus vector of mouse chemokine MIP-1 伪 and costimulatory molecule B7-1 was constructed by using lentivirus as a vector, and its expression in 293T cells was identified, which laid a foundation for the experimental study of double gene therapy for lymphoma. [methods] 1. In the first part, the full-length encoding sequence cDNA, of mouse MIP-1 伪 and B7-1 genes was synthesized by using self-designed primers. The target gene was amplified by nested PCR, and the lentivirus vector was digested by Age I enzyme. The target gene was directly ligated with the lentivirus vector which was linearized by enzyme digestion, and its product was transformed into E. coli DH5 伪 competent cells. The positive clones were identified by PCR and sequenced directly. The result of sequencing is consistent with the target gene sequence, that is, the successful construction of the target gene plasmid. Then the constructed target gene plasmid was extracted by ultrapure endotoxin removal. 2. In the second part, MIP-1 伪 and B7-1 target gene plasmids were transfected into 293T cells respectively. After 48 hours, the expression of green fluorescent protein (GFP) of fluorescent reporter gene was observed, and the expression of the target gene plasmid in 293T cells was detected by Western Blot method. 3. In the third part, pGC-LV recombinant lentivirus vector, packaging plasmid pHelper 1.0 vector and pHelper 2.0 vector were used to co-transfect 293T cells. After 48 hours of culture, supernatants containing lentivirus particles were collected. Concentrated and purified lentivirus concentrate with high titer was obtained. 10 渭 l lentivirus concentrate was transfected into 293T cells, and total RNA, was extracted by reverse transcription to obtain cDNA,. The titer of lentivirus concentrate was detected by real-time fluorescence quantitative PCR. [result] 1. The recombinant lentivirus vector of mouse chemokine MIP-1 伪 and costimulatory molecule B7-1 was successfully constructed. 2. After transfection of MIP-1 伪 and B7-1 target gene plasmids into 293T cells, the expression of MIP-1 伪 and B7-1 target gene plasmids in 293T cells were successfully detected by, Western Blot. 3. The three plasmids were successfully cotransfected into 293T cells and packaged with high titer of MIP-1 伪 and B7-1 recombinant lentivirus concentrate. Real-time quantitative PCR confirmed that MIP-1 伪 and B7-1 recombinant lentivirus vectors all had the titer of 2.00E8 TU/ml.. [conclusion] the recombinant lentivirus vector with high titer of mouse chemokine MIP-1 伪 and costimulatory molecule B7-1 was successfully constructed and packaged in this study, which laid a foundation for the experimental study of double gene therapy for lymphoma.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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