融合型独特型B细胞淋巴瘤DNA疫苗的制备及其免疫学活性测定

发布时间：2018-03-27 08:14

本文选题：B细胞淋巴瘤　切入点：融合型独特型DNA疫苗　出处：《河北医科大学》2008年硕士论文

【摘要】： 目的 B细胞淋巴瘤的膜表面免疫球蛋白(surface membrane immunoglobulin,mIg)独特型(idiotype,Id)来自基因重排,由可变区的重链(VH)和轻链(VL)基因所决定,其突出的个体化特征即肿瘤特异性抗原(TSA)正好满足了抗肿瘤疫苗的特异性的需要。将来源于个体的恶性增殖的B细胞的Ig特异性片段VH和VL通过基因克隆制备成单链抗体可变区片段(scFv),二者由一段短肽连接,可以模拟完整免疫球蛋白的免疫原性。由于scFv源于自身抗原,单独应用不足以诱导抑制肿瘤细胞的免疫反应,为此人们在提高免疫原性方面做了大量的研究。研究者发现利用配体与细胞表面受体的结合可提高抗原呈递给抗原提呈细胞(APC)的效率,提高抗原特异性免疫反应。Biragyn将单核细胞趋化蛋白3(Monocyte chemotactic protein-3,MCP3)与scFv偶联作为疫苗免疫小鼠,发现其表达的融合蛋白具有Ig的天然构象及趋化因子的功能,能和抗原结合,同时吸引抗原提呈细胞(特别是未成熟树突状细胞)向抗原靠近,诱导的免疫反应强于免疫球蛋白疫苗。同时,抗肿瘤的DNA疫苗也是最近研究的一个热点,本研究利用小鼠B细胞淋巴瘤细胞株A20的scFv和MCP3的融合基因构建DNA疫苗,并用该疫苗免疫同源小鼠,LDH法检测小鼠的脾细胞与靶细胞A20孵育后的CTL活性。比较表达scFv和MCP3-scFv的DNA疫苗产生的细胞免疫反应对肿瘤细胞的杀伤效果,探讨融合型独特型DNA疫苗在抗肿瘤免疫反应中是否能诱导T细胞介导的抗肿瘤反应,诱导的抑瘤效果是否高于独特型DNA疫苗,为提高独特型疫苗的免疫原性提供理论基础,为淋巴瘤疫苗的制备及临床试验研究拓宽思路。方法: 1真核表达质粒的构建 1.1 PCR引物设计根据A20细胞IgVH cDNA、IgVL cDNA与MCP3序列分别设计MCP3-scFv和scFv的上下游引物,此部分实验已经完成[1]。根据EGFP序列设计EGFP上下游引物,序列均来源于Genebank。 1.2 PCR扩增目的片段以pGLo/MCP3-scFv为模板,分别用MCP3上游引物和VL下游引物扩增MCP3-scFv,VH上游引物和VL下游引物扩增scFv。以含有EGFP基因序列的质粒PCX-EGFP为模板,分别用引物EGFP上游引物和EGFP下游引物扩增EGFP基因。 1.3 pGEM T-easy载体克隆MCP3-scFv、scFv、EGFP片段以及鉴定 1.4 DNA质粒构建将PCR产物MCP3-scFv、scFv、EGFP酶切后,定向克隆到真核表达载体pTARGET ,得到真核表达质粒pTARGET/EGFP、pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP,将三种质粒转化感受态细菌及酶切鉴定,选取阳性克隆测序。 2质粒转染及鉴定将构建的质粒分别电穿孔法转染至培养至对数生长期的人脐静脉内皮细胞ECV-304,转染后48小时,在倒置荧光显微镜下观察融合蛋白的表达。 3质粒准备和免疫小鼠 3.1质粒纯化应用QIAGEN/Endofree Plasmid Maxi,Giga Kit分离纯化质粒pTARGET/EGFP、pTARGET/scFv-EGFP、pTARGET/MCP3-scFv-EGFP。每种质粒内毒素水平控制在0.1 EU/ug以下,制备成为符合动物实验要求的质粒。 3.2 DNA疫苗免疫动物将18只Babl/c小鼠按随机区组方法分成三组:pTARGET/EGFP组; pTARGET/scFv-EGFP组;pTARGET/MCP3-scFv-EGFP组。6只/组。在小鼠双侧股四头肌肌内注射2.5g/L盐酸布比卡因,每侧50μl。5天后每组质粒取50微克溶至50微升0.9%NaCL分别注射于小鼠两侧的股四头肌,接种三次,每次间隔两周。 4 LDH释放法测定CTL的特异性杀伤率于小鼠免疫结束后第5天取脾细胞作为效应细胞,A20细胞为靶细胞,将效靶比例设置为20/1、50/1、100/1,应用LDH释放法测定CTL活性。 5统计学分析 CTL的杀伤率数据分析采用SPSS(13.0)统计软件进行,3组间比较采用方差分析,两两比较采用SNK-q检验,P0.05认为有统计学意义。结果: 1真核表达质粒经相应的限制性内切酶酶切鉴定均正确。测序结果显示目的片段基因序列与相关文献报道基本符合。 2细胞转染及荧光观察:融合基因真核表达质粒转染ECV后经倒置荧光显微镜下观察发现,pTARGET/EGFP、pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP在转染后48h和72h均表达带较强绿色荧光的蛋白,间接提示融合蛋白成功表达。 3各组CTL杀伤率(%)为:效靶比例为100/1时:pTARGET/MCP3-scFv-EGFP组:68.45±5.72 ; pTARGET/scFv-EGFP组:14.20±4.39 ; pTARGET /EGFP组: 10.76±1.02。pTARGET/MCP3-scFv-EGFP组与其它两组比较均有统计学差异(P0.05)。结论: 1成功构建两种DNA疫苗: pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP。 2 DNA疫苗免疫动物后可诱导出特异性细胞免疫反应。同时试验证明,与scFv疫苗相比,MCP3与scFv融合的独特型DNA疫苗能诱导高效的抗肿瘤免疫应答。
[Abstract]:objective
Surface membrane immunoglobulin B cell lymphoma (surface membrane, immunoglobulin, mIg) (idiotype, Id) unique type from gene rearrangement by the heavy chain variable region (VH) and light chain (VL) determined by individual genes, its prominent characteristic is tumor specific antigen (TSA) just to meet resistance tumor vaccine specific needs. The future source of malignant proliferation in individual B cells Ig specific fragment of VH and VL were prepared by gene clone fragment scFv antibody variable region (scFv), the two are connected by a short peptide, can simulate the complete immunoglobulin scFv due to immunogenicity. Due to the self antigen immune response alone is not sufficient to induce inhibition of tumor cells, so people in to do a lot of research to improve immunogenicity. The researchers found that using a combination of ligands and cell surface receptors can improve the antigens to antigen presenting cells (APC). To improve the efficiency of antigen-specific immune responses to.Biragyn monocyte chemotactic protein 3 (Monocyte chemotactic, protein-3, MCP3) and scFv coupling as vaccine in mice, we found that the expression of the fusion protein has the natural conformation of Ig and chemokines, and antigen binding, while attracting antigen-presenting cells (especially immature dendritic cells antigen) to close, the induced immune response in immunoglobulin vaccine. At the same time, a hot spot of anti-tumor DNA vaccine is a recent research, construct the fusion of mouse B cell lymphoma cell lines A20 scFv and MCP3 gene DNA vaccine used in this study, and the vaccine congenic mice, CTL the activity of mice were detected by LDH spleen cells and target cells incubated with A20. Expression of the lethal effect of cellular immune response to scFv and MCP3-scFv DNA vaccine on tumor cells, to investigate the fusion type Type DNA vaccine is unique in antitumor immunity can induce T cell mediated antitumor response induced by antitumor effect is higher than that of idiotypic DNA vaccine, providing theoretical basis for improving the immunogenicity of idiotype vaccine, for lymphoma vaccine preparation and clinical trial research broaden thinking.
Method:
Construction of 1 eukaryotic expression plasmids
1.1 PCR primer design
According to A20 cell IgVH cDNA, IgVL cDNA and MCP3 sequences, MCP3-scFv and scFv upstream and downstream primers were designed respectively. This part of experiments have been completed. [1]. is designed according to EGFP sequence. Upstream and downstream primers are derived from the EGFP.
1.2 PCR amplification of target fragments
Using pGLo/MCP3-scFv as template, MCP3 upstream primers and VL downstream primers were used to amplify MCP3-scFv. VH upstream primers and VL primers were used to amplify scFv., EGFP PCX-EGFP was used as template, and primer EGFP upstream primers and downstream primers were used to amplify the gene.
1.3 pGEM T-easy vector cloned MCP3-scFv, scFv, EGFP fragment and identification
1.4 DNA plasmid PCR products MCP3-scFv, scFv, EGFP after enzyme digestion and cloned into the eukaryotic expression vector pTARGET and eukaryotic expression plasmid pTARGET/EGFP, pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP, three kinds of plasmids were transformed into competent bacteria and enzyme digestion, selected positive clones were sequenced.
Transfection and identification of 2 plasmids
The plasmids were transfected into human umbilical vein endothelial cells (ECV-304) cultured in logarithmic growth phase by electroporation respectively. After 48 hours of transfection, the expression of fusion protein was observed under inverted fluorescence microscope.
3 plasmids preparation and immunization of mice
3.1 plasmids purification
QIAGEN/Endofree Plasmid Maxi and Giga Kit were used to separate and purify plasmid pTARGET/EGFP, pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP.. Each plasmid's level of endotoxin was controlled below 0.1 EU/ug, and the plasmid was prepared to meet the requirements of animal experiment.
3.2 DNA vaccine for immune animals
18 Babl/c mice were randomly divided into three groups: group pTARGET/EGFP group; pTARGET/scFv-EGFP group; pTARGET/MCP3-scFv-EGFP group.6 / group. Mice in bilateral femoral head four intramuscular injection of 2.5g/L bupivacaine hydrochloride, femoral head four muscle on each side of 50 l.5 days after each take 50 micrograms of plasmid solution were injected into the 0.9%NaCL to 50 L mice on both sides of the three dose, the time interval of two weeks.
The specific killing rate of CTL was determined by 4 LDH release method. Spleen cells were taken as effective cells fifth days after the end of immunization in mice, A20 cells were target cells, and the target target proportion was set to 20/1,50/1100/1. CTL activity was detected by LDH release method.
5 statistical analysis
The killing rate data of CTL were analyzed by SPSS (13) statistical software. The 3 groups were compared by ANOVA, and 22 compared with SNK-q test. P0.05 thought there was statistical significance.
Result:
1 eukaryotic expression plasmids were correctly identified by restriction endonuclease digestion. The sequencing results showed that the sequence of the target fragment was basically consistent with the related literature.
2 cell transfection and fluorescence observation: the fusion gene eukaryotic expression plasmid was transfected into ECV by inverted fluorescence microscope observation, pTARGET/EGFP, with a strong green fluorescence protein expression of pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP in 48h and 72h after transfection, suggesting that indirect fusion protein was successfully expressed.
3, the killing rate (%) of CTL in each group was: the effective target ratio was 100/1: pTARGET/MCP3-scFv-EGFP group: 68.45 + 5.72; pTARGET/scFv-EGFP group: 14.20 + 4.39; pTARGET /EGFP group: 10.76 + 1.02.pTARGET/MCP3-scFv-EGFP group compared with other two groups (P0.05).
Conclusion:
1 successfully constructed two kinds of DNA vaccines: pTARGET/scFv-EGFP and pTARGET/MCP3-scFv-EGFP.
2 DNA vaccine can induce specific cellular immune response after animal immunization. Meanwhile, the test proved that compared with scFv vaccine, the idiotypic DNA vaccine fused with MCP3 and scFv can induce an effective anti-tumor immune response.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R733.1

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