IL-21 gpi和sGM-CSF共同修饰的瘤苗构建及其抗肿瘤效应

目的:构建膜锚定IL-21和分泌性GM-CSF(sGM-CSF)双表达瘤苗,并对其抗肿瘤效应及其机制作初步探讨.方法:用分子生物学方法构建双表达IL-21 gpi和sGM-CSF重组质粒,将鉴定过的重组质粒以脂质体转染B16F10细胞制成瘤苗,用流式细胞仪检测转染瘤苗IL-21 gpi和sGM-CSF的表达.以瘤苗治疗荷瘤鼠,经观察小鼠肿瘤体积、生存率来分析瘤苗的抗瘤性,并检测了瘤苗治疗鼠的细胞免疫活性.结果:正确构建了pRSC/IL-21 gpi-sGM-CSF重组质粒,转染细胞可很好地表达膜锚定IL-21和分泌性GM-CSF,制备的瘤苗能有效地发挥抗肿瘤效应,其机制与瘤苗治疗鼠的脾细胞增殖活性、NK细胞及CD8+细胞细胞毒活性增强有关.结论:成功构建了具有抗肿瘤活性的膜锚定IL-21和分泌性GM-CSF双表达瘤苗,为进一步抗肿瘤免疫治疗研究奠定了基础.     

表达ESAT-6-gpi和IL-21的B16F10瘤苗的构建及其活性鉴定

为构建膜表达糖基化磷脂酰肌醇(GPI)锚定的结核杆菌早期分泌靶抗原6 kD(ESAT-6)和分泌IL-21的B16F10瘤苗并鉴定其活性,利用重叠PCR法构建pIRES-ESAT-6-gpi/IL-21重组质粒,以脂质体转染重组质粒到B16F10细胞,G418筛选出阳性克隆,用RT-PCR、免疫荧光、FCM和Western blot检测瘤苗细胞靶抗原表达,用瘤苗细胞培养上清刺激小鼠CD8+T细胞,检测瘤苗所分泌IL-21的生物学活性。结果表明,pIRES-ESAT-6-gpi/IL-21重组质粒DNA测序正确,B16F10-ESAT-6-gpi/IL-21瘤苗细胞目的基因ESAT-6表达于瘤苗细胞表面,增殖能力未受外源基因导入影响,分泌的IL-21具有生物学活性,为研究膜表达ESAT-6和分泌表达IL-21瘤苗的抗瘤效应奠定了基础。     

B16F10/ESAT-6-GPI-IL-21瘤苗安全性及抗肿瘤效应研究

目的:评估B16F10/ESAT-6-GPI-IL-21瘤苗用于C57BL/6小鼠的安全性及其诱导的抗肿瘤免疫效应。方法:应用免疫荧光、FCM检测瘤苗细胞ESAT-6-GPI的表达情况;以Western blot方法检测瘤苗细胞IL-21的表达情况;动物实验检测瘤苗体内应用的安全性;制备荷瘤鼠术后免疫模型,评价瘤苗免疫效果。结果:B16F10/ESAT-6-GPI-IL-21瘤苗细胞表面有靶抗原ESAT-6-GPI表达,并分泌IL-21。于C57BL/6小鼠皮下接种2×105个B16F10/ESAT-6-GPI-IL-21瘤苗细胞,60天内未见致瘤,但能够诱导小鼠产生有效的抗肿瘤免疫效应。结论:B16F10/ESAT-6-GPI-IL-21瘤苗致瘤性明显下降,低剂量应用具有安全性,能够诱导小鼠产生有效的抗肿瘤免疫效应。     

HSV-2gD核酸疫苗的构建及免疫效果观察

目的:在前期筛选的HSV-2gD模拟抗原表位序列P6的基础上,以pcDNA3.1(-)为载体、IL-18为佐剂,构建P6与IL-18串联的重组表达质粒,并在真核细胞中表达。将重组质粒免疫小鼠,观察免疫效果。方法:采用串联简并引物PCR法构建重组质粒pcDNA3.1-IL18-P6和pcDNA3.1-IL18,以酶切、测序鉴定。将重组质粒分别转染CHO细胞进行瞬时表达,通过间接免疫荧光和Western blot法检测P6的表达情况。将构建的真核表达质粒肌内注射免疫接种BALB/c小鼠3次,每次间隔1周。末次免疫后1周眼眶静脉采血,ELISA法检测小鼠血清特异性抗体滴度、IFN-γ及IL-18含量;末次免疫后1个月,处死小鼠,无菌分离脾脏,MTT法测定脾淋巴细胞增殖率。结果:构建的串联重组质粒pcDNA3.1-IL18-P6和pcDNA3.1-IL18经酶切及测序显示基因序列完全正确,经间接免疫荧光、Western blot法检测可证实模拟抗原表位序列P6具有近似天然序列的生物学活性。重组核酸疫苗pcDNA3.1-IL-18-HSV P6免疫小鼠后可刺激血清特异性抗体的产生,诱导分泌较高水平的IFN-γ和IL-18。可促进小鼠脾淋巴细胞的增殖。结论:成功构建和表达了重组质粒pcD-NA3.1-IL18-P6,其能诱导较强的细胞免疫和体液免疫,从而为构建更加有效的HSV-2DNA疫苗奠定了良好的基础。     

IL-21基因治疗小鼠H22细胞皮下移植肝癌模型的效果评价

用已构建的mIL-21/pcDNA3.1重组质粒对H22细胞建立的小鼠肝癌模型进行基因治疗,观察IL-21对小鼠体内抗肿瘤免疫应答的影响及对小鼠生存的影响。采用BALB/c小鼠左腋皮下注射腹水型肝癌细胞株H22细胞建立小鼠移植肝癌模型,给荷瘤小鼠瘤体内注射mIL-21/pcDNA3.1进行基因治疗,MTT比色法检测IL-21对荷瘤小鼠T细胞增殖水平及NK细胞杀伤活性的影响,观察治疗后荷瘤小鼠生存情况及肿瘤生长情况的改变。病理检测结果显示,成功建立了小鼠移植型肝癌模型,MTT比色法显示基因治疗后小鼠T细胞增殖水平及NK细胞杀伤活性显著升高,荷瘤小鼠肿瘤生长速度减慢,生存期显著延长。IL-21基因治疗肝癌荷瘤小鼠可显著提高荷瘤小鼠体内抗肿瘤免疫应答水平,抑制肿瘤生长,延长荷瘤小鼠生存期。     

小鼠白细胞介素21 cDNA的克隆及真核表达质粒的构建

目的：克隆小鼠自细胞介素2l(IL-21)基因，构建真核表达质粒，用以进行肿瘤的基因治疗。方法：用RT-PCR法。从ConA活化的小鼠T细胞中扩增IL-21 cDNA。克隆人哺乳动物细胞高效表达质粒pcDNA3．1中，构建重组mIL-21真核表达质粒。重组体用载体上的通用引物和PCR下游引物为测序引物，鉴定克隆的正确性。将已鉴定的重组质粒用脂质体法转染Sp2／0细胞，用RT-PCR法鉴定转染细胞中IL-21基因的表达，用MTT比色法检测表达的mIL-21诱导的NK细胞杀伤活性的增强。结果：正确构建了重组真核表达质粒pcDNA3．1／mIL-21，并在转染的细胞中检测出IL-2l的表达，表达的mIL-21可在体外增强NK细胞的杀伤活性：结论：成功地构建了重组真核表达质粒pcDNA3．1／mIL-21，为进一步在肿瘤动物模型中进行IL-21基因治疗及疗效观察奠定了基础.     

pcDNA3.1+/MAGE-3 DNA疫苗诱导特异性抗肿瘤免疫应答的实验研究

目的：构建pcDNA3.1+/MAGE-3 DNA疫苗，观察其在小鼠体内诱导特异性抗肿瘤免疫应答的能力。方法： 通过RT-PCR构建重组表达质粒pcDNA3.1+/MAGE-3；以pcDNA3.1+/MAGE-3 DNA疫苗免疫已接种肿瘤细胞的小鼠，每10 d重复免疫1次，共3次，以pcDNA3.1+、PBS为对照。末次免疫后5 d检测血清中MAGE-3抗体滴度、小鼠脾淋巴细胞的细胞毒T细胞（cytotoxic T lymphocytes，CTL）杀伤活性、细胞因子IL-2和IFN-γ的浓度，同时计算抑瘤率。结果： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，用此疫苗免疫已接种B16/MAGE-3细胞的小鼠后，能诱导小鼠脾淋巴细胞MAGE-3特异性的杀伤活性，脾细胞培养上清中细胞因子IL-2和IFN-γ的浓度明显增高，血清中抗MAGE-3抗体在1∶20滴度时阳性，肿瘤生长被显著抑制，与pcDNA3.1+组、PBS组相比，差异显著（P＜0.01）。结论： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，该疫苗在小鼠体内既能激活CTL杀伤活性和CD4+ T细胞活性，又能激活体液免疫反应，从而诱导出特异性的抗肿瘤免疫应答。     

丙型肝炎病毒核心蛋白DNA免疫初步研究

目的　观察HCV核心蛋白基因的DNA免疫效果。方法　将HCV核心蛋白基因插入真核表达载体pcDNA3.1( ) ,构建重组质粒pcDNA3.1 c。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,用重组质粒 10 0 μg免疫小鼠 ,同时设立空白质粒组和PBS组两组对照 ,初次免疫后 4周、8周各进行一次加强免疫。小鼠体液免疫反应和T淋巴细胞增殖检测分别采用间接免疫荧光法和MTT法。结果　pcDNA3.1 c可在COS7细胞内表达HCV核心抗原 ,接种于Balb c小鼠能有效诱导体液和细胞免疫应答。结论　重组质粒pcDNA3.1 c对于丙型肝炎防治具有潜在价值     

HPV16E7E6抗原表位嵌合体DNA抗肿瘤疫苗的研究

目的:探讨HPV16 E7、E6抗原表位与HSP70 N端重组DNA疫苗抗肿瘤活性.方法:以重叠PCR将HPV16 E7基因N端60个氨基酸的编码序列与E6基因的10个(48～57)氨基酸的编码序列及HSP70 N端序列进行融合.以pcDNA3.0为载体,构建真核表达载体pcD-E76HSP.重组质粒免疫C57BL/6小鼠后,通过淋巴细胞增殖实验和细胞毒性杀伤实验研究该疫苗激发的细胞免疫反应及反应强度;观察该疫苗对C57BL/6小鼠TC-1肿瘤细胞移植瘤的治疗效果.结果:重组DNA疫苗免疫C57BL/6小鼠后,小鼠脾淋巴细胞体外增殖明显,并可诱导产生针对TC-1肿瘤细胞的特异性CTL反应;体内抑瘤试验显示该疫苗对HPV16病毒转化的TC-1细胞小鼠移植瘤的生长有抑制作用.结论:该疫苗能激发特异性细胞免疫反应,显著抑制HPV16转化的TC-1肿瘤细胞生长.     

GPI修饰的ESAT-6核酸疫苗的构建及其免疫功能初探

目的构建糖基化磷脂酰肌醇(GPI)修饰的结核杆菌早期分泌性抗原靶(ESAT-6)核酸疫苗,初步分析其免疫功能。方法利用重叠PCR法构建pIRES-ESAT-6-gpi重组体,转染B16F10细胞,G418筛选阳性克隆,用RT-PCR、免疫荧光检测转染细胞的ESAT-6抗原表达情况;采集ESAT-6核酸疫苗免疫鼠血清,分别检测抗体滴度和CDC效应,免疫磁珠法分选CD4+和CD8+T细胞,CFSE/7-AAD细胞毒实验分析CTL细胞毒活性。结果测序正确的重组体pIRES-ESAT-6-gpi转染细胞后,ESAT-6表达于细胞膜表面。ESAT-6核酸疫苗免疫血清和CD8+T细胞分别通过CDC效应和细胞毒作用杀伤膜表达ESAT-6的B16F10细胞。结论构建的GPI修饰的ESAT-6核酸疫苗能够诱导免疫鼠产生体液和细胞免疫反应,杀伤膜表达ESAT-6的B16F10细胞,为进一步基于该法构建B16F10瘤苗的抗肿瘤免疫效应及机制研究奠定了基础。     

mIL-21基因治疗小鼠肿瘤及其机制的初步研究

建立小鼠Sp2/0皮下肿瘤模型,以我们构建并鉴定过的真核表达质粒peDNA3.1/mIL-21在瘤体内直接注射,考察mIL-21基因治疗对小鼠Sp2/0肿瘤的疗效。结果显示pcDNA3.1/mIL-21质粒治疗对小鼠皮下Sp2/0肿瘤有一定的生长抑制作用,CTL和NK细胞毒活性明显增强,IFN-γ分泌升高显著,但抗体无明显升高。表明mIL-21基因治疗的抗肿瘤效应,可能和细胞免疫功能增强有关。     

Preliminary study on mouse interleukin-21 application in tumor gene therapy

lnterleukin-21(IL-21)is a recently characterized T cell-derived cytokine with a significant homology to IL-2,IL-4 and IL-15.To determine whether IL-21 has broad immunoregulatory activity and can stimulate durableantitumour responses,we constructed mouse IL-21(mIL-21) recombinant plasmid and evaluated its antitumorefficacy.Mouse IL-21 cDNA was amplified from Con A-activated mouse T cells by RT-PCR.RecombinantpcDNA3.1/mIL-21 was constructed and transfected into Sp2/0 cells.Mouse IL-21 expression was analyzed byWestern blotting and its activities were detected by ~3H-TdR incorporation and MTT assay.The recombinantpcDNA3.1/mIL-21 was injected s.c.into tumor lump.Tumor size,weight,the activities of CTLs,NK cells andLAK cells and serum IFN-γ,level were measured for evaluating mIL-21 mediated antitumor responses.Theresults indicated that mIL-21 was correctly expressed in Sp2/0 cells and it can improve the proliferation of Tcells and B cells,and enhance NK cytotoxic activity in vitro.The activities of CTL and NK cells,and serumIFN-γlevel were significantly improved,furthermore the tumor growth was obviously suppressed inpcDNA3.1/mIL-21 treated mice.However,the LAK activity did not alter significantly.Taken together,thisstudy suggests that the injection with recombinant plasmid containing mIL-21 is a potential approach fortumor gene therapy.Cellular & Molecular Immunology.2004;1(6):461-466.     

Expression, purification and identification of recombinant mouse interleukin 21 protein in E. coli

Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.     

嗜肺军团菌免疫原蛋白核酸疫苗诱导的小鼠免疫应答及其保护力

目的:研究嗜肺军团菌免疫原蛋白核酸疫苗诱导的小鼠免疫原性以及对LP感染小鼠的保护能力。方法:用嗜肺军团菌免疫原蛋白基因真核表达重组质粒pcDNA3.1-ip作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFNγ-产生水平和CTL特异杀伤活性等指标,以评价疫苗的免疫原性。真核表达重组质粒pcDNA3.1-ipDNA疫苗重复免疫BALB/c小鼠2次,末次免疫2周后,用10倍LD50剂量攻击小鼠,计数小鼠的存活数及小鼠肺中的细菌数,观察感染鼠的肺部病理变化。结果:pcDNA3.1-ip免疫小鼠后诱导产生了特异的体液免疫应答和细胞免疫应答,免疫组的免疫原性和免疫保护性均高于对照组pcDNA3.1( )组(P<0.01)。结论:免疫原蛋白基因可作为嗜肺军团菌核酸疫苗的侯选基因。     

Comparison of immune responses induced in mice by vaccination with DNA vaccine constructs expressing mycobacterial antigen 85A and interleukin-21 and Bacillus Galmette-Guérin

In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.     

Interleukin-12 gene modification exerts anti-tumor effects on murine mammary sarcoma cell line in vivo

The aim of this project was to investigate the anti-tumor effect of an IL-12 gene modified mammary sarcoma murine cell line, EMT6/IL-12, in mouse model. In this study, we transfected the recombinant eukaryotic plasmid encoding IL-12 gene （pcDNA6-p70） into EMT6 and obtained the IL-12 expressing EMT6/IL-12 cell line. Then EMT6/IL-12 cells were s.c. inoculated into mice. The recombinant vector treatment group was set as control. We then evaluated the inhibition of tumor growth and the anti-tumor immunity function in vivo such as cytotoxicity, proliferation of splenocytes and serial IFN-T level. And the percentage of IFN-T producing CD4 or CD8 T cells among splenocytes was also analyzed in tumor bearing mice. Our results showed that the growth of tumors was obviously inhibited in EMT6/IL-12 group. Moreover, the capacities of anti-tumor immunity were all significantly higher in EMT6/IL-12 group compared to the controls. The results of the present investigation support the notion that EMT6/IL-12 could exert gene therapy in tumor model by improving the anti-tumor cellular immunity.     

IL-23、IL-27对RSV重组蛋白疫苗免疫原性的影响

目的:以编码IL-23和IL-27的真核表达质粒pcDNA3.1-IL-23(以下简称IL-23)和pcDNA3.1-IL-27(以下简称IL-27)为佐剂,与呼吸道合胞病毒(RSV)重组蛋白疫苗G1F/M2共免疫小鼠,观察IL-23和IL-27对疫苗的免疫原性的影响。方法:以IL-23、IL-27质粒和Al(OH)3为免疫佐剂,与G1F/M2共免疫BALB/c小鼠,末次免疫后10天杀死小鼠,用ELISA检测特异性IgG、IgG1和IgG2a水平;流式细胞术检测小鼠脾细胞CD4+、CD8+细胞的变化;用乳酸脱氢酶(LDH)释放法检测特异性小鼠脾细胞杀伤活性。结果:G1F/M2+Al(OH)3+IL-23和G1F/M2+Al(OH)3+IL-27可诱导高效价的IgG、IgG1、IgG2a抗体,且显著高于这三种佐剂单独使用诱导的抗体效价;流式细胞检测结果显示G1F/M2+IL-23和G1F/M2+Al(OH)3+IL-23刺激的CD4+T细胞和CD8+T细胞水平显著高于其他组;单独的IL-23或IL-27对G1F/M2诱导的脾细胞杀伤活性没有增强作用,而Al(OH)3+IL-23和Al(OH)3+IL-27佐剂组的杀伤率显著高于单独的IL-23、IL-27或Al(OH)3组。结论:这些结果表明:IL-23或IL-27质粒与传统铝盐佐剂Al(OH)3联合使用能显著增强RSV重组疫苗G1F/M2的免疫原性。     

弓形虫多表位DNA疫苗的构建及其免疫保护作用

目的:构建弓形虫多表位DNA疫苗并研究其免疫保护效果.方法:将编码含弓形虫多个T、B细胞表位的6段弓形虫多肽基因,以5个甘氨酸编码基因相间隔相连接,克隆入真核表达质粒pcDNA3.1( )中,构建成多表位弓形虫DNA疫苗.免疫BALB/c小鼠,测定其诱导的特异性抗体水平及T淋巴细胞增殖状况,同时进行弓形虫攻击感染保护实验.结果:成功构建了包含多个弓形虫表位的真核表达质粒pcDNA3.1/T-ME,以其作为DNA疫苗免疫小鼠,可诱导机体产生弓形虫特异性的体液及细胞免疫应答,产生有效的抗弓形虫保护性免疫应答.结论:构建的弓形虫多表位DNA疫苗能诱导机体产生有效的保护性免疫应答,在控制弓形虫感染上具有可行性.     

过继肿瘤特异性淋巴细胞联合mIL-21瘤苗抗肿瘤效应研究

目的 在环磷酰胺(Cy)所致小鼠淋巴细胞急剧减少状态下,过继mIL-21转染的瘤苗细胞(mIL-21-Sp2/0)免疫致敏的同系小鼠淋巴细胞,联合mIL-21-Sp2/0瘤苗免疫,探讨过继免疫抗瘤效应机制.方法 用Cy 100 mg/kg预处理BALB/c小鼠2 d后,以灭活mIL-21瘤苗免疫的同系小鼠脾及淋巴结的淋巴细胞作为肿瘤抗原特异性淋巴细胞过继给Cy预处理鼠,同时用mIL-21瘤苗免疫,7 d后以野生型Sp2/0细胞攻击,观察小鼠的肿瘤生长情况.流式细胞仪(FCM)分析CD4+、CD8+、CD4+CD25+T细胞等亚群的变化,分别以CFSE和7-AAD标记,FCM检测淋巴细胞增殖能力和效应细胞的细胞毒活性,用ELISPOT检测分泌IFN-γ的淋巴细胞数量.结果 与对照组相比,Cy预处理小鼠接受过继效应细胞及mIL-21瘤苗免疫后,可有效地抵抗野生型Sp2/0细胞的攻击.过继的肿瘤特异性淋巴细胞的体内增殖能力和体外杀伤活性显著增强,分泌IFN-γ效应细胞的数量显著增高.结论 在小鼠淋巴细胞减少期,过继mIL-21瘤苗免疫致敏的肿瘤抗原特异性淋巴细胞并同时给予mIL-21瘤苗免疫,利于输入的效应细胞及自身免疫细胞的增殖和特异性抗肿瘤功能的形成与维持.