PSCA-HSP70融合蛋白可溶性表达的研究及小鼠前列腺肿瘤模型的建立

发布时间：2018-01-18 01:36

本文关键词：PSCA-HSP70融合蛋白可溶性表达的研究及小鼠前列腺肿瘤模型的建立　出处：《中国人民解放军军事医学科学院》2008年硕士论文　论文类型：学位论文

更多相关文章： PSCA HSP70 前列腺癌 荧光素酶

【摘要】： 前列腺干细胞抗原(Prostate stem cell antigen, PSCA)为含有123个氨基酸的糖蛋白,属于Thy-1/Ly-6家族GPI锚定的细胞表面抗原。研究表明,PSCA在正常前列腺组织中低表达,而在膀胱癌、胰腺癌及雄激素依赖性和非依赖性前列腺癌组织中高表达;而且有研究表明,抗PSCA的抗体能够在体内抑制肿瘤的生长。以上研究为以PSCA为靶点的新型疫苗的研制奠定基础。如能以PSCA作为靶点,辅以合适的佐剂分子,利用基因工程技术获得重组蛋白疫苗,则有可能对相关的肿瘤起到积极的治疗作用。但是在本课题前期的研究中发现,PSCA蛋白难以表达,或是表达的PSCA蛋白可溶性很低,以包涵体的形式存在,且蛋白复性困难,使得疫苗研制难度加大,不利于PSCA蛋白疫苗的构建及免疫活性的研究。分析其原因,可能是由于PSCA为膜蛋白,本身结构复杂,含有多个半胱氨酸,不易折叠为正确的高级结构。因此,如何增强PSCA的可溶性表达,是当前研究的关键;同时,为评价重组蛋白的免疫效果,需要建立合适的小鼠前列腺肿瘤模型。本研究选取人源的热休克蛋白70(Heat shock protein70, HSP70)作为佐剂分子,将HSP70与PSCA基因进行融合表达,希望能够利用HSP70分子的佐剂功能和分子伴侣功能,同时增强PSCA蛋白的可溶性和免疫后机体的免疫水平,以构建用于治疗前列腺癌的重组蛋白疫苗。通过对PSCA及HSP70进行结构分析,我们构建了不同形式的融合基因,探索何种融合形式能够促进PSCA的可溶性表达,同时能够降低HSP70的抗原性,并保持HSP70增强抗原免疫效果的活性。首先扩增PSCA及HSP70基因,构建HSP70与PSCA融合基因。通过对PSCA进行结构分析,我们保留其活性结构域的部分即PSCA(T),然后将PSCA(T)、PSCA全长(FL)、HSP70基因分别克隆至pET21a(+)质粒中,构建重组质粒pET21-PSCA(T)-HSP、pET21-PSCA(FL)-HSP、pET21-HSP-PSCA(T)、pET21-HSP-PSCA(FL)。SDS-PAGE电泳分析表明PSCA(T)-HSP在E.coli中得到部分可溶性表达、HSP-PSCA(T)、HSP-PSCA(FL)大多为包涵体,而PSCA(FL)-HSP未得到表达;为了能够降低HSP70的抗原性并保持HSP70增强抗原免疫效果的活性,按结构域将HSP70分为HSPI及HSPII ,分别与PSCA(T)构建重组质粒pET21-PSCA(T)-HSPI、pET21-PSCA(T)-HSPII。SDS-PAGE电泳分析表明两者在E.coli中均得到可溶性表达。本研究最终拟选择PSCA(T)-HSP、PSCA(T)-HSPI及PSCA(T)-HSPII作为前列腺癌治疗性疫苗的候选研究对象。为了评价PSCA重组蛋白的免疫效果,我们建立了C57BL/6小鼠传统前列腺肿瘤动物模型。将小鼠前列腺来源的RM-1细胞稳定转染真核表达质粒pcDNA-PSCA,将筛选得到的稳定表达人PSCA基因的RM-PSCA细胞接种C57BL/6小鼠,结果表明实验小鼠全部成瘤,并且肿瘤生长迅速,小鼠平均存活37天,成功建立了表达人PSCA的小鼠肿瘤动物模型。然而由于传统肿瘤动物模型不能观察体内肿瘤的生长、转移情况;需要靠不同的时间侵入性地观察或宰杀动物以获得实验数据,不能反复跟踪研究;无法反映基因的时间性及空间性表达,为此我们建立了C57BL/6小鼠荧光素酶前列腺肿瘤动物模型,以更加方便、准确评价重组蛋白的免疫效果。为建立C57BL/6小鼠荧光素酶前列腺肿瘤动物模型,我们将真核表达质粒pcDNA-PSCA、pcDNA-Luc稳定共转染RM-1细胞,将筛选得到的稳定表达Luc及人PSCA基因的RM-PSCA/Luc细胞接种C57BL/6小鼠,建立荧光素酶小鼠动物模型。结果表明实验小鼠全部成瘤,肿瘤生长迅速,小鼠平均存活38天,转荧光素酶基因肿瘤细胞生物特性稳定,活体成像仪检测到转染荧光素酶细胞在小鼠体内活体成像,并且荧光素酶表达活性的强弱能够反映肿瘤大小;同时荧光素酶的表达并没有改变肿瘤的生长特性。本模型的建立为今后疫苗的研制奠定基础。
[Abstract]:Prostate stem cell antigen (Prostate stem cell antigen, PSCA) is a glycoprotein containing 123 amino acids, which belongs to the family of cell surface antigen Thy-1/Ly-6 GPI anchored. The study shows that the low expression of PSCA in normal prostate tissue, and in bladder cancer, pancreatic cancer and high expression of androgen dependent and non dependent prostate cancer tissues; but studies have shown that anti PSCA antibody can inhibit tumor growth in vivo. The above research lays the foundation for the development of new vaccines targeting PSCA. As to PSCA as a target, with appropriate adjuvant molecules, the recombinant protein vaccine by genetic engineering, is likely to positive effect related to the tumor. But found in our previous study, PSCA protein is difficult to express, or PSCA soluble protein expression is very low, in the form of inclusion bodies, and protein refolding The vaccine development difficulties, difficulty in study is not conducive to building a PSCA protein vaccine and immune activity. The analysis of its causes, may be due to the PSCA membrane protein, complex structure, containing a plurality of cysteine, not easily folded into advanced structure correctly. Therefore, how to enhance the soluble expression of PSCA, is the key to research; at the same time, in order to evaluate the immune effect of the recombinant protein, we need to establish appropriate mouse models of prostate cancer.
This study selected the human heat shock protein 70 (HSP70 Heat shock protein70) as adjuvant molecules, HSP70 and PSCA gene fusion expression, hoping to use HSP70 molecular adjuvant and chaperone function, and enhance the body's immune level and soluble immune PSCA protein, to build for the treatment of prostate cancer the recombinant protein vaccine. By analyzing the structure of PSCA and HSP70, we constructed a fusion gene in different forms, explore what form of fusion can promote the expression of soluble PSCA, also can reduce the antigenicity of HSP70, and keep the immune enhancement of HSP70 antigen activity.
The amplification of PSCA gene and HSP70 gene, construct HSP70 and PSCA fusion gene. By analyzing the structure of PSCA, we retain the active domain of the part is PSCA (T), and PSCA (T), PSCA (FL), the full-length HSP70 gene was cloned into pET21a (+) plasmid, recombinant plasmid pET21-PSCA (T) -HSP, pET21-PSCA (FL) -HSP, pET21-HSP-PSCA (T), pET21-HSP-PSCA (FL).SDS-PAGE electrophoresis analysis showed that PSCA (T) -HSP was partially soluble expression in E.coli, HSP-PSCA (T), HSP-PSCA (FL) are mostly as inclusion bodies, and PSCA (FL) -HSP is not expressed in order to reduce; the antigenicity of HSP70 and maintain the immune enhancement of HSP70 antigen activity, according to the domain of HSP70 is divided into HSPI and HSPII, respectively, and PSCA (T) to construct the recombinant plasmid pET21-PSCA (T) -HSPI, pET21-PSCA (T) -HSPII.SDS-PAGE electrophoresis analysis showed that the two in E.coli were soluble expression. This study intends to end PSCA (T) -HSP, PSCA (T) -HSPI and PSCA (T) -HSPII are selected as candidates for therapeutic vaccines for prostate cancer.
In order to evaluate the immune effect of recombinant PSCA protein, we established C57BL/6 mice traditional prostate tumor animal model. The mice prostate derived RM-1 cells stably transfected with eukaryotic expression plasmid pcDNA-PSCA, obtained the stable expression of human PSCA gene in RM-PSCA cells inoculated into C57BL/6 mice showed all the mice tumor, and tumor growth rapidly. The mice survived 37 days on average, successfully established a mouse tumor animal model of human PSCA expression.
However due to the traditional model of tumor animal observation of tumor growth and metastasis; need to rely on different time to observe the invasive or to kill an animal to obtain experimental data, can not be repeated tracking; can not reflect the expression of time and space genes, we established mouse C57BL/6 luciferase prostate tumor animal model, with more convenient, accurate evaluation of the immune effect of the recombinant protein.
For the establishment of C57BL/6 mouse luciferase prostate tumor animal model, we will eukaryotic expression plasmid pcDNA-PSCA pcDNA-Luc were transfected into RM-1 cells, obtained stable expression of Luc and PSCA gene in RM-PSCA/Luc cells inoculated into C57BL/6 mice to establish animal model of small rat luciferase. The results showed that all the mice tumor, the tumor grew rapidly, the average of mice live for 38 days, the biological characteristics of tumor cells to the luciferase gene stable in vivo imaging was detected by cell transfection in vivo in mice in vivo imaging and expression of luciferase activity intensity can reflect the growth characteristics and tumor size; luciferase expression did not alter the tumor. This model lays the foundation for future vaccine development.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R737.25;R-332

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