以前列腺干细胞抗原为靶点的重组蛋白及重组腺病毒载体疫苗的研究
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摘要
基于本课题前期关于以PSCA(Prostate stem cell antigen, PSCA)为靶点的前列腺癌治疗性DNA疫苗的研究结果,本研究拟选取人源的热休克蛋白70(Heat shock protein70,HSP70)作为佐剂分子,构建用于前列腺癌治疗用的重组亚单位疫苗及重组腺病毒载体疫苗,期望能够利用HSP70分子的佐剂功能和分子伴侣功能增强PSCA蛋白的可溶性和免疫后机体的免疫水平;同时基于“prime-boost”免疫策略的考虑,希望不同疫苗的联合免疫能够明显提高机体的免疫水平。通过在小鼠前列腺癌模型上系统地检测不同疫苗的免疫水平,评价以PSCA作为治疗靶点的疫苗的免疫效果,为前列腺肿瘤疫苗的研制及临床研究奠定基础。
为构建重组亚单位疫苗,通过对PSCA进行结构分析,保留其活性结构域的部分,然后将PSCA、HSP70基因分别克隆至原核表达载体pET21a(+)质粒中,构建重组表达质粒pET21-PSCA-HSP。SDS-PAGE电泳分析表明PSCA-HSP在E.coli中得到可溶性表达。为了评价重组亚单位疫苗的免疫效果,在大肠杆菌系统中表达了PSCA、HSP70及PSCA-HSP重组蛋白,并经过色谱层析技术获得了具有一定纯度的重组蛋白。免疫C57BL/6雄性小鼠后,通过上述技术方法检测了针对PSCA的细胞免疫和体液免疫水平,并观察小鼠的肿瘤生长情况及死亡时间。研究结果表明,免疫重组蛋白PSCA-HSP能够激起PSCA特异的细胞免疫反应;PSCA、PSCA+HSP及PSCA-HSP免疫组均能诱导机体产生较高的针对PSCA的抗体,三组之间的抗PSCA抗体水平没有差异;HSP、PSCA+HSP及PSCA-HSP免疫组均诱导机体产生了较高水平的针对HSP的抗体,三组之间的抗体水平没有差异;免疫PSCA、PSCA+HSP及PSCA-HSP均能抑制小鼠肿瘤的生长,延长荷瘤小鼠的存活时间,其中PSCA-HSP的抗肿瘤效果要优于其它两组。
为构建重组腺病毒载体疫苗,将PSCA基因从实验室保存的质粒pcDNA-PSCA中酶切获得,将PSCA-HSP融合基因从实验室保存的质粒pcDNA-PSCA-HSP中酶切获得,分别克隆至pDC316质粒中,构建重组穿梭质粒pDC316-PSCA及pDC316-PSCA-HSP,将重组穿梭质粒进行腺病毒包装,制备重组腺病毒Ad5-PSCA及Ad5-PSCA-HSP。为了评价重组腺病毒载体疫苗的免疫效果,免疫C57BL/6雄性小鼠后,通过ELISPOT、胞内细胞因子检测和ELISA等手段检测了针对PSCA的细胞免疫和体液免疫水平,并观察小鼠的肿瘤生长情况及死亡时间。研究结果表明,免疫Ad5-PSCA及Ad5-PSCA-HSP均能够激起机体强烈的PSCA特异的细胞免疫反应,但是各组针对PSCA的抗体效价水平均较低;免疫Ad5-PSCA及Ad5-PSCA-HSP均能抑制小鼠肿瘤的生长,延长荷瘤小鼠的存活时间,其中Ad5-PSCA-HSP的抗肿瘤效果要优于Ad5-PSCA。
基于“prime-boost”免疫策略,本研究将以PSCA为靶点的重组腺病毒载体疫苗以及重组亚单位疫苗联合免疫C57BL/6雄性小鼠,通过上述各项免疫指标的检测比较不同疫苗的细胞及体液免疫水平,并观察小鼠的肿瘤生长情况及死亡时间。研究结果表明,各免疫组均能激起PSCA特异的细胞及体液免疫反应;各免疫组均能抑制小鼠肿瘤的生长,延长荷瘤小鼠的存活时间,其中Ad5-PSCA-HSP+PSCA-HSP混合免疫方式获得了最佳的抗肿瘤效果。
本研究构建了基于PSCA为靶点的重组腺病毒载体疫苗及重组亚单位疫苗,并在小鼠前列腺肿瘤模型上评价了不同疫苗的免疫效果,为前列腺肿瘤疫苗的研制及临床研究奠定基础。
In view of the prophase study of therapeutic DNA vaccine for prostate cancer based on PSCA, in this study, HSP70 was chosen as adjuvant molecule to be coupled with PSCA to construct recombinant protein vaccine and recombinant adenovirus vector vaccine for prostate cancer treatment, expecting to increase the soluble expression of PSCA with the activity of enhancing the immune response. Furthermore, combined vaccines were inoculated to C57BL/6 mice on account of the immune strategy of "prime-boost". The immune responses and therapeutic efficacy of these vaccines based on PSCA were evaluated in mice model, which lays the foundation for development of vaccines for prostate cancer and clinical research.
For construction of recombinant protein vaccine, the PSCA and HSP70 genes were amplified by PCR. PSCA, only containing nucleotides encoding the active structural domain, was cloned into prokaryote expression vector pET21a(+) with HSP70 gene to construct recombinant plasmid pET21-PSCA-HSP. PSCA-HSP was confirmed to be expressed in soluble condition in E.coli by SDS-PAGE electrophoresis analysis. For evaluating the immune effect of the recombinant protein vaccine, PSCA、HSP70 and PSCA-HSP were expressed in E.coli system and purified with chromatography methods. After immunization with these recombinant protein, the PSCA-specific cellular and humoral immune responses were detected by using ELISPOT assay, intracellular cytokines staining assay and ELISA assay. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with recombinant protein PSCA-HSP could induce a PSCA-specific cellular immune response. Meanwhile, the mice vaccinated with recombinant protein PSCA+HSP、PSCA and PSCA-HSP could induce a PSCA-specific humoral immune response. They all generated high level of anti-PSCA antibody without difference. Furthermore, recombinant protein PSCA+HSP、PSCA and PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the mice’s survival. Meanwhile, PSCA-HSP could obtain the optima antitumous effect.
After being obtained from pcDNA-PSCA plasmid and pcDNA-PSCA-HSP plasmid by digesting with restriction enzyme, PSCA gene and fusion gene PSCA-HSP were separately cloned into pDC316 shuttle vector to construct recombinant shuttle plasmids pDC316-PSCA and pDC316-PSCA-HSP. Recombinant shuttle plasmids were packed with recombinant adenovirus, then recombinant Ad5-PSCA and Ad5-PSCA-HSP were prepared. For evaluating the immune effect of recombinant adenovirus vector vaccine, ELISPOT assay, intracellular cytokines staining assay and ELISA assay were used to detect the PSCA-specific cellular and humoral immune responses. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with Ad5-PSCA and Ad5-PSCA-HSP both could induce a strong PSCA-specific cellular immune response. However, the level of anti-PSCA antibody was low among these groups. Furthermore, they both could inhibit the growth of PSCA-expressing tumors and prolong the mice’s survival. Meanwhile, Ad5-PSCA-HSP could obtain the optima antitumous effect.
On account of the immune strategy of "prime-boost", in this study, recombinant protein vaccine and recombinant adenovirus vector vaccine were inoculated to C57BL/6 mice respectively. The PSCA-specific cellular and humoral immune responses were detected by using ELISPOT assay, intracellular cytokines staining assay and ELISA assay. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with vaccines could induce a PSCA-specific cellular and humoral immune response. Furthermore, all these vaccines could inhibit the growth of PSCA-expressing tumors and prolong the mice’s survival. Meanwhile, Ad5-PSCA-HSP+PSCA-HSP could obtain the optima antitumous effect.
In this study, the recombinant Ad5-PSCA-HSP and the recombinant protein PSCA-HSP were successfully constructed. Cellular and humoral immune responses induced by different vaccines were evaluated in mice, which lays the foundation for development of vaccines for prostate cancer and clinical research.
为构建重组亚单位疫苗,通过对PSCA进行结构分析,保留其活性结构域的部分,然后将PSCA、HSP70基因分别克隆至原核表达载体pET21a(+)质粒中,构建重组表达质粒pET21-PSCA-HSP。SDS-PAGE电泳分析表明PSCA-HSP在E.coli中得到可溶性表达。为了评价重组亚单位疫苗的免疫效果,在大肠杆菌系统中表达了PSCA、HSP70及PSCA-HSP重组蛋白,并经过色谱层析技术获得了具有一定纯度的重组蛋白。免疫C57BL/6雄性小鼠后,通过上述技术方法检测了针对PSCA的细胞免疫和体液免疫水平,并观察小鼠的肿瘤生长情况及死亡时间。研究结果表明,免疫重组蛋白PSCA-HSP能够激起PSCA特异的细胞免疫反应;PSCA、PSCA+HSP及PSCA-HSP免疫组均能诱导机体产生较高的针对PSCA的抗体,三组之间的抗PSCA抗体水平没有差异;HSP、PSCA+HSP及PSCA-HSP免疫组均诱导机体产生了较高水平的针对HSP的抗体,三组之间的抗体水平没有差异;免疫PSCA、PSCA+HSP及PSCA-HSP均能抑制小鼠肿瘤的生长,延长荷瘤小鼠的存活时间,其中PSCA-HSP的抗肿瘤效果要优于其它两组。
为构建重组腺病毒载体疫苗,将PSCA基因从实验室保存的质粒pcDNA-PSCA中酶切获得,将PSCA-HSP融合基因从实验室保存的质粒pcDNA-PSCA-HSP中酶切获得,分别克隆至pDC316质粒中,构建重组穿梭质粒pDC316-PSCA及pDC316-PSCA-HSP,将重组穿梭质粒进行腺病毒包装,制备重组腺病毒Ad5-PSCA及Ad5-PSCA-HSP。为了评价重组腺病毒载体疫苗的免疫效果,免疫C57BL/6雄性小鼠后,通过ELISPOT、胞内细胞因子检测和ELISA等手段检测了针对PSCA的细胞免疫和体液免疫水平,并观察小鼠的肿瘤生长情况及死亡时间。研究结果表明,免疫Ad5-PSCA及Ad5-PSCA-HSP均能够激起机体强烈的PSCA特异的细胞免疫反应,但是各组针对PSCA的抗体效价水平均较低;免疫Ad5-PSCA及Ad5-PSCA-HSP均能抑制小鼠肿瘤的生长,延长荷瘤小鼠的存活时间,其中Ad5-PSCA-HSP的抗肿瘤效果要优于Ad5-PSCA。
基于“prime-boost”免疫策略,本研究将以PSCA为靶点的重组腺病毒载体疫苗以及重组亚单位疫苗联合免疫C57BL/6雄性小鼠,通过上述各项免疫指标的检测比较不同疫苗的细胞及体液免疫水平,并观察小鼠的肿瘤生长情况及死亡时间。研究结果表明,各免疫组均能激起PSCA特异的细胞及体液免疫反应;各免疫组均能抑制小鼠肿瘤的生长,延长荷瘤小鼠的存活时间,其中Ad5-PSCA-HSP+PSCA-HSP混合免疫方式获得了最佳的抗肿瘤效果。
本研究构建了基于PSCA为靶点的重组腺病毒载体疫苗及重组亚单位疫苗,并在小鼠前列腺肿瘤模型上评价了不同疫苗的免疫效果,为前列腺肿瘤疫苗的研制及临床研究奠定基础。
In view of the prophase study of therapeutic DNA vaccine for prostate cancer based on PSCA, in this study, HSP70 was chosen as adjuvant molecule to be coupled with PSCA to construct recombinant protein vaccine and recombinant adenovirus vector vaccine for prostate cancer treatment, expecting to increase the soluble expression of PSCA with the activity of enhancing the immune response. Furthermore, combined vaccines were inoculated to C57BL/6 mice on account of the immune strategy of "prime-boost". The immune responses and therapeutic efficacy of these vaccines based on PSCA were evaluated in mice model, which lays the foundation for development of vaccines for prostate cancer and clinical research.
For construction of recombinant protein vaccine, the PSCA and HSP70 genes were amplified by PCR. PSCA, only containing nucleotides encoding the active structural domain, was cloned into prokaryote expression vector pET21a(+) with HSP70 gene to construct recombinant plasmid pET21-PSCA-HSP. PSCA-HSP was confirmed to be expressed in soluble condition in E.coli by SDS-PAGE electrophoresis analysis. For evaluating the immune effect of the recombinant protein vaccine, PSCA、HSP70 and PSCA-HSP were expressed in E.coli system and purified with chromatography methods. After immunization with these recombinant protein, the PSCA-specific cellular and humoral immune responses were detected by using ELISPOT assay, intracellular cytokines staining assay and ELISA assay. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with recombinant protein PSCA-HSP could induce a PSCA-specific cellular immune response. Meanwhile, the mice vaccinated with recombinant protein PSCA+HSP、PSCA and PSCA-HSP could induce a PSCA-specific humoral immune response. They all generated high level of anti-PSCA antibody without difference. Furthermore, recombinant protein PSCA+HSP、PSCA and PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the mice’s survival. Meanwhile, PSCA-HSP could obtain the optima antitumous effect.
After being obtained from pcDNA-PSCA plasmid and pcDNA-PSCA-HSP plasmid by digesting with restriction enzyme, PSCA gene and fusion gene PSCA-HSP were separately cloned into pDC316 shuttle vector to construct recombinant shuttle plasmids pDC316-PSCA and pDC316-PSCA-HSP. Recombinant shuttle plasmids were packed with recombinant adenovirus, then recombinant Ad5-PSCA and Ad5-PSCA-HSP were prepared. For evaluating the immune effect of recombinant adenovirus vector vaccine, ELISPOT assay, intracellular cytokines staining assay and ELISA assay were used to detect the PSCA-specific cellular and humoral immune responses. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with Ad5-PSCA and Ad5-PSCA-HSP both could induce a strong PSCA-specific cellular immune response. However, the level of anti-PSCA antibody was low among these groups. Furthermore, they both could inhibit the growth of PSCA-expressing tumors and prolong the mice’s survival. Meanwhile, Ad5-PSCA-HSP could obtain the optima antitumous effect.
On account of the immune strategy of "prime-boost", in this study, recombinant protein vaccine and recombinant adenovirus vector vaccine were inoculated to C57BL/6 mice respectively. The PSCA-specific cellular and humoral immune responses were detected by using ELISPOT assay, intracellular cytokines staining assay and ELISA assay. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with vaccines could induce a PSCA-specific cellular and humoral immune response. Furthermore, all these vaccines could inhibit the growth of PSCA-expressing tumors and prolong the mice’s survival. Meanwhile, Ad5-PSCA-HSP+PSCA-HSP could obtain the optima antitumous effect.
In this study, the recombinant Ad5-PSCA-HSP and the recombinant protein PSCA-HSP were successfully constructed. Cellular and humoral immune responses induced by different vaccines were evaluated in mice, which lays the foundation for development of vaccines for prostate cancer and clinical research.
引文
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1.Kim JJ,Trivedi NN,Wilson DM,et al.Molecular and immunological analysis of genetic prostate specific antigen(PSA) vaccine.Oncogene,1998,17:3125-3135.
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49.任军,李建民,陈薇.联合疫苗应用现状及评价.中国生物工程杂志,2009,29(11):89-93.
1.Kim JJ,Trivedi NN,Wilson DM,et al.Molecular and immunological analysis of genetic prostate specific antigen(PSA) vaccine.Oncogene,1998,17:3125-3135.
2.Elagib KE,Xiao M,Hussaini,IM,et al.Jun blockade of erythropoiesis:role for repression of GATA-1 by HERP2.Mol Cell Biol,2004,24(17):7779-7794.
3.Zlotocha S,Staab MJ,Horvath D,et al.A phase I study of a DNA vaccine targeting prostatic Acid phosphatage in patients with stage DO prostate cancer.Clinical genitourinary cancer,2005,4:215-218.
4.Dannull J ,diener PA ,Prikler L ,et al. Prostate stem cell antigen is a promising candidate for immunotherapy of advanced prostate cancer.Cancer Res,2000,60(19):5522-5528.
5.Noda S ,Kosugi A ,Saitoh S ,et al. Protection from anti-TCR/ CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2. J Exp Med,1996,183(5):2355-2360.
6.Reiter RE ,Gu z ,Watabe T ,et al. Prostate stem cell antigen :a cellsurface maker overexpressed in prostate cancer . Natl Acad Sci USA ,1998,95(4):1735-1740.
7.Antica M ,Wu L ,Scollay R. Stem cell antigen 2 expression in adult and developing mice.Immunol Lett ,1997,55(1):47-51.
8.Nakamura K,Yasunaga Y,Ko D,et al.Cadmium-induced plastic transformation of human prostate epithelial cells.lnt J Oncol,2002,20(3);543-547.
9.Gu Z , Thomas G, Yamashiro J , et al. Prostate stem cell antigen (PSCA) expression increases with high gleason score ,advanced stage and bone metastasis in prostate cancer[J].Oncogene,2000,19 (10):1288-1296.
10.Saffran DC ,Raitano AB ,Hubert RS ,et al.Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts. Proc Natl Acad Sci USA,2001,98(5):2658-2663.
11.Ross S ,Spencer SD ,Holcomb I ,et al. Prostate stem cell antigen as therapy target:tissue expression and in vivo efficacy of an immunoconjugate. Cancer Res ,2002 ,62 ,2546-2553.
12.Zhang XP ,Yu CG ,Zhao J ,et al. Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen-specfic CD8+ T-cell response and inhibits the PSCA+ tumors growth in mice [J]. J Gene Med ,2007 ,9:715-726.
13.Maria de la Luz Garcia-Hernandez ,Andrew Gray ,Bolyn Hubby , et al. Prostate Stem Cell Antigen Vaccination Induces a Long-term Protective Immune Response against Prostate Cancer in the Absence of Autoimmunity. Cancer Res ,2008 ,68:(3): 861-869.
14.S Kim ,JB Lee ,GK Lee ,et al.Vaccination with recombinant adenoviruses and dentritic cells expressing prostate-specific antigens is effective in eliciting CTL and suppresses tumor growth in the exprerimental proatate cancer.Prostate ,2009 ,69(9):938-948.