基因免疫研究进展

1990年WolffJA等意外发现小鼠骨骼肌细胞能直接摄取病毒裸露的DNA ,并表达其编码的蛋白质 ,这种表达可持续数月 ,这一发现为开创基因疫苗的研究奠定了基础。 1992年Tang等将带有人生长激素的质粒DNA通过基因枪导入小鼠表皮细胞 ,几周后 88%被接种的小鼠产生了特异性抗体。 1993年Ulmer等发现小鼠肌肉注射编码甲型流感病毒相对保守的核心蛋白的质粒载体后 ,可有效的保护小鼠抵抗另一亚型流感病毒的攻击 ,从而证明了注射不但能在体内表达抗原蛋白 ,并且可以诱导产生具有显著保护作用的免疫应答。从此继全菌弱毒苗和亚单位疫苗后 ,出现了…     

弓形虫SAG1,ROP1基因及其复合抗原基因的核酸免疫研究

目的 检测混合SAG1和ROP1编码基因真核表达质粒疫苗诱导小鼠的免疫应答,评价其抗弓形虫感染的保护性免疫效果.方法 将SAG1和编码基因片段克隆入pEGFP-N3表达载体,构建重组质粒;RT-PCR体外验证重组质粒在NH3T3细胞中的表达;通过检测抗体、抗体分型及细胞因子来评价体液免疫和细胞免疫;腹腔内注射毒性株弓形虫速殖子攻击免疫小鼠.结果 RT-PCR结果显示重组质粒能在哺乳动物细胞内表达;SAG1和ROP1混合重组质粒疫苗诱导小鼠产生很强的体液免疫和细胞免疫,对于毒性株弓形虫感染攻击具有保护作用.结论 不同候选抗原编码基因重组质粒能够诱导小鼠产生抗弓形虫感染保护性免疫,提示含有多种成分的混合DNA疫苗的研制可作为核酸免疫研究的策略之一.     

gD2 DNA疫苗诱导细胞免疫应答

用生殖器疱疹gD2 DNA疫苗pcDNA3-gD转染哺乳动物细胞COS-7，鉴定gD表达．再用其免疫BALB／c小鼠，测定脾细胞增殖、CTL细胞杀伤活性，以及CD4^ 和CD8^ 细胞群．结果表明：pcDNA3-gD能够在哺乳动物细胞COS-7中表达gD抗原，其免疫小鼠脾细胞增殖活性增加，CTL活性达37．69％，明显高于pcDNA3组和生理盐水组；CD4^ 占T细胞数量的33．38％，数量较pcDNA3组和生理盐水组显著增加．     

弓形虫pcDNA3-ROP1 DNA疫苗免疫小鼠诱导体液免疫应答及保护性的初步观察

目的 观察弓形虫pcDNA3-ROP1 DNA疫苗免疫BALB/C小鼠诱导其体内产生的体液免疫应答及抗虫感染的免疫保护作用.方法 重组质粒用生理盐水稀释后,肌注免疫BALB/C小鼠.分别于免疫后5周和10周用ELISA法测定IgG抗体滴度;免疫鼠腹腔注射弓形虫速殖子攻击感染.结果 经两次检测,均可测到特异性IgG抗体,且抗体的滴度随免疫时间的延长而增高;弓形虫速殖子腹腔攻击感染免疫组小鼠的平均存活时间较对照组延长,统计学有显著性差异(P<0.01).结论 弓形虫pcDNA3-ROP1质粒 DNA疫苗能诱导BALB/C小鼠产生特异性体液免疫应答及部分抗虫免疫保护作用.     

日本血吸虫Sj23与IL-12多价DNA疫苗的构建

构建能共同表达日本血吸虫23kDa膜抗原(Sj23)与细胞因子IL-12的多价DNA疫苗．RT-PCR的方法从日本血吸虫成虫获得Sj23的基因片段，克隆到载体pVIVO2-mIL12／mcs上，进行酶切和测序鉴定．采用免疫荧光的方法检测多价DNA疫苗pVIVO2-Sj23-IL12在小鼠骨骼肌细胞的表达．成功地构建了能共同表达日本血吸虫23kDa膜抗原(Sj23)与细胞因子IL-12的多价DNA疫苗．此多价DNA疫苗在免疫小鼠肌细胞的细胞膜和细胞浆均获得了Sj23的表达．     

基于数字疫苗的隐遁勒索病毒攻击动态防御模型

针对隐遁勒索病毒攻击威胁性极大以及传统方法对其防御不力的问题,该文提出了一种基于数字疫苗的隐遁勒索病毒攻击动态防御模型。借鉴生物免疫机理,给出了数字疫苗、抗原、抗体及抗体浓度等免疫概念的形式化定义。首先,通过接种数字疫苗(创建诱饵文件和文件夹),使系统生成抵御隐遁勒索病毒攻击的未成熟抗体;其次,通过免疫抗体动态演化机制,生成能抵御隐遁勒索病毒抗原的成熟抗体与记忆抗体;最后,通过在内核层和应用层实施双重动态监控抗体浓度变化,并借助交叉视图法来实时感知隐遁勒索病毒攻击。理论分析与实验结果表明:该模型有效解决了隐遁勒索病毒攻击难以实时检测的问题,且较传统方法性能更高。     

结核分支杆菌Ag85B DNA疫苗构建、免疫原性及其保护效力

构建结核分支杆菌分泌蛋白Ag85B编码基因的DNA疫苗,研究其免疫原性和保护效力.构建该DNA疫苗并经鉴定后,应用Qiagen试剂盒大量纯化无内毒素的质粒DNA.将C57BL/6小鼠随机分为4组,分别3周间隔3次用生理盐水（A）、载体质粒（B）、卡介苗（C）、Ag85B DNA疫苗（D）免疫小鼠,通过ELISA方法检测血清中抗Ag85B抗体及其亚型.第三次免疫后28 d用结核分支杆菌H37Rv攻击,观察小鼠体重变化,攻击4周后检测细胞因子IFN-γ、IL-12,进行肺组织病理检查及菌落计数等.经Ag85B DNA疫苗免疫的小鼠特异性抗体IgG均显著高于对照组,而且IgG2b均高于IgG1和IgG2a.结核分支杆菌攻击后各组小鼠体重变化呈现不同趋势,A、B组小鼠体重从第3周开始下降;C组体重总体呈上升趋势;D组体重第2周开始下降,之后维持不变.MTT法检测脾淋巴细胞增殖实验中D组的刺激指数显著高于其它组;D组小鼠脾淋巴细胞转化率也显著高于A、B组.肺组织菌落计数C组最低,其次是D组.大体病理结果显示C组和D组肺组织病变较A、B组轻.A、B组肺泡腔内有较多渗出液,肺泡壁充血、水肿.C组肉芽肿数目最多.上述结果表明Ag85B DNA疫苗具有较强的免疫原性,呈TH1型细胞介导的免疫应答.Ag85B DNA疫苗的免疫保护效力不如BCG,有待进一步研究其应用价值.     

AsiaⅠ型口蹄疫病毒基因工程多肽疫苗的构建及其免疫原性分析

为了构建Asia Ⅰ型口蹄疫病毒多肽疫苗,用反转录PCR方法扩增VPI基因并测得该基因的核苷酸序列．将VPI（133AA-163AA）、VP2（1AA～33AA）抗原表位基因分别与卢一半乳糖苷酶基因（简称LacZ＇）3’末端相连构建表达质粒LacZ’-VIP和LacZ’-VP2．化学合成VP1（133AA～163AA）-VP2（1AA～33AA）-VPl（133AA～163AA）串联重复抗原表位DNA,然后分别连接在β-半乳糖苷酶基因和IgG重链恒定区基因的3’末端,构建两种重组表达质粒LacZ’-VPl（133AA--163AA）-VP）2（1AA～33AA）-VP1（133AA-163AA）（命名为pTl）和IgGvPl（133AA～163AA）-VP2（1AA～33AA）-VPl（133AA-163AA）（命名为pT2）．通过Westernblot、血清中和抗体效价测定、T细胞增殖反应及豚鼠抗病毒保护实验分析所构建疫苗的免疫原性及有效性．结果显示pT1、pT2表达的融合蛋白能够诱发豚鼠产生较高的中和抗体及刺激豚鼠T细胞增殖．pT1、pT2两种融合蛋白分别2次免疫豚鼠后,100％的豚鼠能抵抗100 ID50 AsiaⅠ型口蹄疫病毒攻击．pT2融合蛋白1次免疫豚鼠后70％的豚鼠能抵抗100 ID50 AsiaⅠ型口蹄疫病毒攻击．     

重组百日咳杆菌粘附素C端蛋白的免疫原性

分别将支气管败血波氏杆菌百日咳杆菌粘附素(PRN)基因prn的全长编码区2 040 bp及3'端867 bp的片段(prnC)克隆到原核表达载体pGEX-KG,经IPTG诱导后在大肠杆菌中实现了表达.Western blot检测证实两种表达产物GST-PC和GST-PRN均具有良好的免疫学反应活性.在主动免疫保护试验中,GST-PC和GST-PRN免疫组小鼠均能产生较高的PRN抗体水平;当使用3 LD50的Bb强毒株HH0809进行鼻腔攻毒后,其保护率均为100% (9/9);当使用10 LD50 HH0809攻毒时,其保护率分别为66.7% (6/9)和77.8% (7/9).在被动免疫保护试验中,腹腔免疫GST-PC和GST-PRN的兔抗血清均能100% (10/10)保护小鼠抵抗10 LD50的HH0809的腹腔攻击,但经PRN吸附后的2种兔抗血清均失去了保护力(0/5).研究结果表明:重组PRN C端多肽具有良好的免疫原性,可考虑作为新型亚单位疫苗或活载体疫苗的抗原成分.     

小鼠结核分枝杆菌感染模型用于评价重组HBHA疫苗免疫效果的实验研究

目的通过复制小鼠结核病模型评价重组HBHA疫苗的免疫效果。方法将重组肝素结合血凝素(HBHA)疫苗接种BALB/c小鼠,观察其诱导的细胞免疫应答水平,并以MTB H37Rv毒株攻击免疫小鼠,研究重组疫苗诱导的保护力。结果重组HBHA免疫BALB/c小鼠后可诱发细胞毒作用,将体内MTB裂解。重组疫苗不仅能刺激CD4+T,CD8+T增殖、活化,还可诱导脾细胞分泌IFN-γ、IL-2等细胞因子。H37Rv毒株攻击后,被免疫小鼠的组织病理学症状减轻。结论本研究通过检测小鼠体内细胞免疫应答和肺脏组织病理学改变等指标,客观的反映了重组疫苗免疫小鼠结核模型的保护效果。     

Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

肝片吸虫DNA疫苗的构建及其免疫效应的初步研究

用ＥcorRI酶切含肝片吸虫保护性抗原基因ＦＨ３的重组质粒pUC18/FH3,回收ＦＨ３（－１．０Ｋb)片段克隆到真核表达载体pBlueCMV上,酶切筛选顺向插入重组子pBlueCMV-FH3,即得到一种肝片吸虫ＤＮＡ疫苗,制备纯化该疫苗并免疫小鼠,小鼠肌肉细胞中有一定的ＦＨ３抗原表达;用ＥＬＩＳＡ检测抗血清表明,注射疫苗的小鼠均伴随产生一定量的特异性抗体,用肝片吸早囊蚴攻击（２０囊蚴／鼠）小鼠,初步显示有一定的减虫率。     

用于基因疫苗接种的气动式基因枪

为解决现有各种基因枪在微弹制备方面存在的问题并减小冲击波影响,提出一种基因枪结构。将裹着基因疫苗的微弹吸附在不锈钢网上,并将不锈钢网放置在加速通道中加速微弹。在数值模拟的基础上,研制了实验装置,测试了装置的性能,并利用该装置将pEGFG质粒导入H e la细胞。实验结果表明:这种结构的基因枪在0.3M Pa的气压下就可以把微弹加速到300m/s以上,且微弹在靶面分布均匀;射入H e la细胞的pEGFG质粒也得到了表达,能满足基因疫苗接种的要求。     

HIV-1C亚型DNA疫苗诱导小鼠免疫反应的研究

 构建含中国流行株HIV-1 C亚型核心蛋白gag基因的重组质粒pVAX-gag,并在体外进行了表达与鉴定.同时构建了含此gag基因的原核表达质粒pGEX-gag,表达纯化并鉴定重组蛋白Gag.以质粒pVAX-gag免疫Balb/C小鼠后,用ELISpot和流式细胞仪检测其细胞免疫反应.再以纯化后的重组蛋白Gag作为包被抗原,用ELISA检测其体液免疫反应.结果显示重组质粒pVAX-gag免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应,且免疫剂量和免疫效果存在一定的正相关性.重组原核表达质粒pGEX-gag的表达产物能与抗p24单克隆抗体发生特异性反应,可用于抗HIV抗体检测.     

Specific anti-tumor effect induced by attenuated <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1＋ VEGFR2（n1-7） was transformed into competent attenuated Salmonella typhimuriurn SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1＋VEGFR2（n1-7）. To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy （TEM） was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an enhanced accumulation of CD8^＋ cytotoxic T lymphocytes, as well as an increase in CD4^＋ cells in the tumore of animals treated with the oral gene vaccine compared to tumors from control group mice. UI- trestructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the r     

一种新型基因枪的研究

为研制一种适应各种场合 ,尤其是在野外操作的基因枪 ,满足将目的基因导入动植物细胞 ,使之稳定表达并遗传 ,实现培育或改良新的优良品种。以压缩弹簧为动力 ,运用撞击发射原理 ,对裹着基因的微粒进行加速 ,使之获得足够的动能 ,去射击靶细胞。弹簧预压力不同 ,微弹的动能随之不同 ,可适应不同细胞的要求。利用所设计的装置 ,在常压下将GUS基因导入番茄叶片细胞 ,经检测 ,得到表达 ;将质粒psv- luc2 0转入小鼠的皮肤、腹部肌肉和肝脏内 ,也得到表达。实验表明 ,该构思是可行的 ,实验装置可以在常压下操作 ,能在各种条件下使用     

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

0 IntroductionInbroicguhltat einxpge cDtaNtiAonvafcocri nper eivse ant ninegw aanpdp cruoraicnhg wdiits-heases . Due to the comparative weakness of cellulari mmunity that is purely induced by E7 DNA vac-cine,research on DNA vaccine is now focused onhowto enhance the i mmunogenicity of DNA vac-cine.It has been found in some research that thestrength of cellular i mmunity activity has things todo with the speed of antigen molecules’degrada-tion,i .e.the faster the degradation,the higher th…     

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

OBJECTIVE: To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines. METHODS: BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA; splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro. RESULTS: The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone, but there was not significantly different (P>0.05). Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-Gamma in supernatant of splenocytes cul-tured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05). CONCLUSION: The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.     

乙肝病毒核酸疫苗诱导小鼠细胞免疫应答的研究

研究利用核酸疫苗诱生乙型肝炎病毒 (HBV)表面抗原 (S)细胞免疫应答的作用 .免疫前于BALB/C小鼠股四头肌注射 75 %盐酸布比卡因 ,三天后同样部位注射包含HBsAg基因片段的重组质粒pcDNAHBs .采用3H -TdR掺入法测定免疫小鼠脾细胞增殖能力 ;XTT法测定其脾细胞分泌IL 2活性 .结果表明注射乙肝核酸疫苗可以使小鼠脾细胞增殖 ,小鼠脾细胞分泌上清中可检测到IL - 2活性