本文關(guān)鍵詞：PSCA-HSP70融合蛋白可溶性表達(dá)的研究及小鼠前列腺腫瘤模型的建立　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： PSCA HSP70 前列腺癌 熒光素酶

【摘要】： 前列腺干細(xì)胞抗原(Prostate stem cell antigen, PSCA)為含有123個(gè)氨基酸的糖蛋白,屬于Thy-1/Ly-6家族GPI錨定的細(xì)胞表面抗原。研究表明,PSCA在正常前列腺組織中低表達(dá),而在膀胱癌、胰腺癌及雄激素依賴性和非依賴性前列腺癌組織中高表達(dá);而且有研究表明,抗PSCA的抗體能夠在體內(nèi)抑制腫瘤的生長(zhǎng)。以上研究為以PSCA為靶點(diǎn)的新型疫苗的研制奠定基礎(chǔ)。如能以PSCA作為靶點(diǎn),輔以合適的佐劑分子,利用基因工程技術(shù)獲得重組蛋白疫苗,則有可能對(duì)相關(guān)的腫瘤起到積極的治療作用。但是在本課題前期的研究中發(fā)現(xiàn),PSCA蛋白難以表達(dá),或是表達(dá)的PSCA蛋白可溶性很低,以包涵體的形式存在,且蛋白復(fù)性困難,使得疫苗研制難度加大,不利于PSCA蛋白疫苗的構(gòu)建及免疫活性的研究。分析其原因,可能是由于PSCA為膜蛋白,本身結(jié)構(gòu)復(fù)雜,含有多個(gè)半胱氨酸,不易折疊為正確的高級(jí)結(jié)構(gòu)。因此,如何增強(qiáng)PSCA的可溶性表達(dá),是當(dāng)前研究的關(guān)鍵;同時(shí),為評(píng)價(jià)重組蛋白的免疫效果,需要建立合適的小鼠前列腺腫瘤模型。 本研究選取人源的熱休克蛋白70(Heat shock protein70, HSP70)作為佐劑分子,將HSP70與PSCA基因進(jìn)行融合表達(dá),希望能夠利用HSP70分子的佐劑功能和分子伴侶功能,同時(shí)增強(qiáng)PSCA蛋白的可溶性和免疫后機(jī)體的免疫水平,以構(gòu)建用于治療前列腺癌的重組蛋白疫苗。通過(guò)對(duì)PSCA及HSP70進(jìn)行結(jié)構(gòu)分析,我們構(gòu)建了不同形式的融合基因,探索何種融合形式能夠促進(jìn)PSCA的可溶性表達(dá),同時(shí)能夠降低HSP70的抗原性,并保持HSP70增強(qiáng)抗原免疫效果的活性。 首先擴(kuò)增PSCA及HSP70基因,構(gòu)建HSP70與PSCA融合基因。通過(guò)對(duì)PSCA進(jìn)行結(jié)構(gòu)分析,我們保留其活性結(jié)構(gòu)域的部分即PSCA(T),然后將PSCA(T)、PSCA全長(zhǎng)(FL)、HSP70基因分別克隆至pET21a(+)質(zhì)粒中,構(gòu)建重組質(zhì)粒pET21-PSCA(T)-HSP、pET21-PSCA(FL)-HSP、pET21-HSP-PSCA(T)、pET21-HSP-PSCA(FL)。SDS-PAGE電泳分析表明PSCA(T)-HSP在E.coli中得到部分可溶性表達(dá)、HSP-PSCA(T)、HSP-PSCA(FL)大多為包涵體,而PSCA(FL)-HSP未得到表達(dá);為了能夠降低HSP70的抗原性并保持HSP70增強(qiáng)抗原免疫效果的活性,按結(jié)構(gòu)域?qū)SP70分為HSPI及HSPII ,分別與PSCA(T)構(gòu)建重組質(zhì)粒pET21-PSCA(T)-HSPI、pET21-PSCA(T)-HSPII。SDS-PAGE電泳分析表明兩者在E.coli中均得到可溶性表達(dá)。本研究最終擬選擇PSCA(T)-HSP、PSCA(T)-HSPI及PSCA(T)-HSPII作為前列腺癌治療性疫苗的候選研究對(duì)象。 為了評(píng)價(jià)PSCA重組蛋白的免疫效果,我們建立了C57BL/6小鼠傳統(tǒng)前列腺腫瘤動(dòng)物模型。將小鼠前列腺來(lái)源的RM-1細(xì)胞穩(wěn)定轉(zhuǎn)染真核表達(dá)質(zhì)粒pcDNA-PSCA,將篩選得到的穩(wěn)定表達(dá)人PSCA基因的RM-PSCA細(xì)胞接種C57BL/6小鼠,結(jié)果表明實(shí)驗(yàn)小鼠全部成瘤,并且腫瘤生長(zhǎng)迅速,小鼠平均存活37天,成功建立了表達(dá)人PSCA的小鼠腫瘤動(dòng)物模型。 然而由于傳統(tǒng)腫瘤動(dòng)物模型不能觀察體內(nèi)腫瘤的生長(zhǎng)、轉(zhuǎn)移情況;需要靠不同的時(shí)間侵入性地觀察或宰殺動(dòng)物以獲得實(shí)驗(yàn)數(shù)據(jù),不能反復(fù)跟蹤研究;無(wú)法反映基因的時(shí)間性及空間性表達(dá),為此我們建立了C57BL/6小鼠熒光素酶前列腺腫瘤動(dòng)物模型,以更加方便、準(zhǔn)確評(píng)價(jià)重組蛋白的免疫效果。 為建立C57BL/6小鼠熒光素酶前列腺腫瘤動(dòng)物模型,我們將真核表達(dá)質(zhì)粒pcDNA-PSCA、pcDNA-Luc穩(wěn)定共轉(zhuǎn)染RM-1細(xì)胞,將篩選得到的穩(wěn)定表達(dá)Luc及人PSCA基因的RM-PSCA/Luc細(xì)胞接種C57BL/6小鼠,建立熒光素酶小鼠動(dòng)物模型。結(jié)果表明實(shí)驗(yàn)小鼠全部成瘤,腫瘤生長(zhǎng)迅速,小鼠平均存活38天,轉(zhuǎn)熒光素酶基因腫瘤細(xì)胞生物特性穩(wěn)定,活體成像儀檢測(cè)到轉(zhuǎn)染熒光素酶細(xì)胞在小鼠體內(nèi)活體成像,并且熒光素酶表達(dá)活性的強(qiáng)弱能夠反映腫瘤大小;同時(shí)熒光素酶的表達(dá)并沒有改變腫瘤的生長(zhǎng)特性。本模型的建立為今后疫苗的研制奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R737.25;R-332

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本文編號(hào)：1438905