汉坦病毒核蛋白基因疫苗的初步研究

探讨了汉坦病毒核蛋白(NP)S基因疫苗的免疫效果。构建重组真核表达质粒pcDNA3.1 S,直接肌注免疫BALB/c小鼠,分别用ELISA法检测血清特异性抗体的变化,MTT法检测T细胞增殖反应,以及用ELISAkit检测免疫鼠脾细胞上清液中细胞因子IL-4和IFN-γ的动态变化,从而了解免疫鼠的体液免疫反应和细胞免疫反应。pcDNA3.1 S接种组的小鼠血清抗体水平、T细胞增殖反应以及细胞因子IL-4和IFN-γ的产生水平均较对照组pcDNA3.1 空载体接种组的要高。编码核蛋白的S基因的核酸免疫可同时启动机体的细胞免疫和体液免疫反应,S基因是汉坦病毒核酸疫苗的一个比较理想的侯选抗原基因。     

人乳头瘤病毒16型E5与IL-12联合基因疫苗的免疫活性

为了研制人乳头瘤病毒16型(HPV16)防治性疫苗,分析了HPV16 E5与IL-12联合基因疫苗的免疫活性。将构建的pcDNA3.1(+)/E5与pcDNA3.1(+)/IL-12联合免疫BALB/c小鼠,以ELISA测定小鼠血清中抗HPV16 E5 IgG水平、小鼠脾细胞培养上清中IFN-γ和IL-4含量;MTT法检测脾淋巴细胞增殖反应。结果显示末次免疫后,联合基因疫苗组和单基因疫苗组血清IgG A450值分别明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);且联合基因疫苗组显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组的IFN-γ和IL-4含量分别均明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组IFN-γ和IL-4含量(P<0.01),且联合基因疫苗组含量显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组脾淋巴细胞刺激指数(SI)分别显著高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);联合基因疫苗组与单基因疫苗组比较,SI差异无统计学意义(P>0.05)。结果表明HPV16 E5单基因疫苗以及与IL-12联合基因疫苗均能刺激机体产生较强的免疫应答,且联合基因疫苗优于单基因疫苗。     

人乳头瘤病毒16型E7变异株免疫原性研究

采用分子克隆技术,构建E7变异株重组质粒[pcDNA3.1-(by)E7]和E7标准株重组质粒[pcDNA3.1-(ys)-E7],并将两种质粒分别皮下免疫Balb/c小鼠,免疫后于不同时间提取小鼠血清和制备脾淋巴细胞悬液,分别用ELISA法和MTT比色法检测特异性抗体和特异性淋巴细胞增殖反应.基因免疫后,ELISA法显示,HPV16 E7变异株和标准株均能诱导特异性抗E7抗体;MTT比色法显示,E7标准株免疫组脾淋巴细胞在体外受到变异株E7蛋白的再次刺激后出现特异性淋巴细胞增殖反应,变异株E7免疫组脾淋巴细胞经过同样处理后,出现非特异性淋巴细胞增殖反应.结果表明HPV16E7变异株能诱导特异性体液免疫应答而不能诱导特异性细胞免疫应答,HPV16 E7变异株无论在结构还是免疫原性上均与标准株有差异.由此推测,HPV16E7变异可能导致其逃逸机体自然感染或疫苗诱导的免疫应答.用基因免疫方法研究E7变异株免疫原性也为其它不能或难以进行体外培养的病毒变异研究提供借鉴.     

HIV-1结构蛋白gag-gp120与白细胞介素2联合基因免疫

在真核表达载体pVAX1中的CMV启动子下游插入IL-2基因,构建真核表达质粒pVAXIL2.将它与表达I型人免疫缺陷病毒(Human immunodeficiency virus 1, HIV-1) gag-gp120的核酸疫苗质粒pVAXGE共同肌肉注射BALB/c小鼠,免疫3次后,以ELISA法检测免疫小鼠血清中抗HIV-1抗体水平,结果显示联合免疫组小鼠在免疫2周后已有抗体产生,6周后进入高峰.乳酸脱氢酶释放法检测免疫小鼠脾特异性CTL杀伤活性,结果显示联合免疫组小鼠脾特异性CTL杀伤活性显著高于pVAXGE单独免疫组(P<0.05)和载体质粒pVAX1对照组(P<0.01).以上结果表明HIV-1核酸疫苗质粒pVAXGE与真核表达质粒pVAXIL2联合免疫可诱导特异性体液免疫和细胞免疫应答,且免疫应答水平高于pVAXGE单独免疫组,IL-2发挥了免疫佐剂的作用,增强了核酸疫苗的免疫原性.     

HIV-1结构蛋白gag-gp120与白细胞介素2联合基因免疫

在真核表达载体pVAX1中的CMV启动子下游插入IL-2基因,构建真核表达质粒pVAXIL2。将它与表达I型人免疫缺陷病毒(Humanimmunodeficiencyvirus1,HIV-1)gag-gp120的核酸疫苗质粒pVAXGE共同肌肉注射BALB/c小鼠,免疫3次后,以ELISA法检测免疫小鼠血清中抗HIV-1抗体水平,结果显示联合免疫组小鼠在免疫2周后已有抗体产生,6周后进入高峰。乳酸脱氢酶释放法检测免疫小鼠脾特异性CTL杀伤活性,结果显示联合免疫组小鼠脾特异性CTL杀伤活性显著高于pVAXGE单独免疫组(P<0.05)和载体质粒pVAX1对照组(P<0.01)。以上结果表明:HIV-1核酸疫苗质粒pVAXGE与真核表达质粒pVAXIL2联合免疫可诱导特异性体液免疫和细胞免疫应答,且免疫应答水平高于pVAXGE单独免疫组,IL-2发挥了免疫佐剂的作用,增强了核酸疫苗的免疫原性。     

HPV6L1/CtMOMP多表位融合基因在COS-7细胞的表达及其在小鼠的免疫效果观察

研究人乳头瘤病毒(human papillomavirus,HPV)6b结构蛋白L1(HPV6b L1)基因佐剂增强沙眼衣原体(Chlamydia trachomatis,Ct)主要外膜蛋白(MOMP)多表位(Ct MOMP168)DNA疫苗的免疫效果的可能性.构建pcDNA3.1(+)/HPV6b L1/Ct MOMP168融合重组质粒,转染COS-7细胞,用RT-PCR、激光共聚焦显微技术及Western印迹技术检测其表达.分别用pcDNA3.1(+)/HPV6b L1/Ct MOMP168、pcDNA3.1(+)/Ct MOMP168及pcDNA3.1(+)质粒肌肉免疫BALB/c小鼠,ELISA检测外周血中IgG及阴道分泌物中sIgA.结果表明,pcDNA3.1(+)/HPV6b L1/CtMOMP168可在COS-7细胞中表达;Ct MOMP168组和HPV6b L1/Ct MOMP168组均可刺激小鼠产生抗Ct MOMP特异性的抗体,抗体滴度随免疫次数增加而升高,且HPV6b L1/Ct MOMP168组小鼠产生的抗体滴度明显高于Ct MOMP168组(P0.05).结果提示,分子佐剂HPV6b L1与CtMOMP多表位基因融合能够显著增强Ct MOMP多表位DNA疫苗的体液免疫应答.     

羊痘病毒P32基因真核表达载体的构建、表达及其免疫原性

通过PCR方法扩增全长P32基因和截去跨膜区的P32基因(MP32),将其分别克隆到真核表达载体pcDNA3.1( )和已插入CpG序列的pcDNA3.1-CpG中,构建pcDNA3.1-P32、pcDNA3.1-CpG-P32和pcDNA3.1-CpG-MP32质粒;用脂质体法转染BHK-21细胞,通过间接免疫荧光(IFA)试验验证其表达效果;经肌肉免疫注射健康BALB/c小鼠,用间接ELISA法检测抗体;在免疫后的第3、5周取免疫小鼠的脾细胞,用流式细胞仪检测CD4 和CD8 T细胞亚群.结果所构建的真核表达载体在BHK-21细胞中都能表达P32蛋白;免疫小鼠血清在免疫第2周后均能检测到羊痘特异性IgG抗体;免疫组小鼠脾脏CD4 T细胞数目和CD4 /CD8 T细胞比值明显高于对照组.结果提示,所构建的真核载体可诱导小鼠产生特异性体液免疫应答,并能刺激小鼠产生较强的细胞免疫应答.     

SARS病毒s1基因片段真核表达载体的构建及免疫效果

本研究根据GenBank登录的BJ01株SARS-CoV序列合成801bpS1基因片段,该片段被亚克隆至真核表达载体pcDNA3.1(+)得到重组质粒pcDNA3.1(+)/S1;转染Hela细胞,SDS-PAGE、Western-Blotting鉴定蛋白表达;肌注免疫BALB/c小鼠,利用ELISA法检测免疫后小鼠的抗SARS/CoVIgG及IFN-γ水平,MTT法检测T细胞增殖活性.结果显示,重组质粒pcDNA3.1(+)/S1可在Hela细胞内表达S1蛋白,免疫后小鼠的T细胞增殖活性增强,抗SARS-CoVIgG与IFN-γ水平升高.本实验说明pcDNA3.1(+)/S1可诱导小鼠产生一定的体液免疫和细胞免疫应答.     

人乳头瘤病毒l6型E6-E7融合蛋白真核表达载体的构建与鉴定

目的构建人乳头瘤病毒l6型(HPV16)E6-E7融合蛋白真核表达载体,为研究其基因疫苗免疫活性奠定实验基础。方法 PCR扩增HPV16 E6-E7基因片段,将其连接到真核表达载体pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)/HPV16 E6-E7,双酶切及测序鉴定。将质粒转染HeLa细胞,RT-PCR鉴定E6-E7基因在HeLa细胞中的表达。提取质粒免疫小鼠,利用免疫组化方法检测在其肌肉组织中的表达。结果成功构建了真核表达载体pcDNA3.1(+)/HPV16 E6-E7;在转染pcDNA3.1(+)/HPV16 E6-E7的细胞中检测到HPV16 E6-E7基因。在免疫该质粒的小鼠肌肉组织中可以检测到该质粒的蛋白表达。结论成功的构建的了真核表达载体pcDNA3.1(+)/HPV16 E6-E7,该载体能在HeLa细胞内以及小鼠骨骼肌细胞内有效表达。     

人乳头瘤病毒16型E7变异株免疫原性研究

采用分子克隆技术,构建E7变异株重组质粒[pcDNA3．1-(by)E7]和E7标准株重组质粒【pcDNA3．1．(ys)-E7】,并将两种质粒分别皮下免疫Balb／c小鼠,免疫后于不同时间提取小鼠血清和制备脾淋巴细胞悬液,分别用ELISA法和MTT比色法检测特异性抗体和特异性淋巴细胞增殖反应。基因免疫后,ELISA法显示,HPVl6E7变异株和标准株均能诱导特异性抗E7抗体;MTTT比色法显示,E7标准株免疫组脾淋巴细胞在体外受到变异株E7蛋白的再次刺激后出现特异性淋巴细胞增殖反应,变异株E7免疫组脾淋巴细胞经过同样处理后,出现非特异性淋巴细胞增殖反应。结果表明HPV16E7变异株能诱导特异性体液免疫应答而不能诱导特异性细胞免疫应答,HPVl6E7变异株无论在结构还是免疫原性上均与标准株有差异。由此推测,HPV16E7变异可能导致其逃逸机体自然感染或疫苗诱导的免疫应答。用基因免疫方法研究E7变异株免疫原性也为其它不能或难以进行体外培养的病毒变异研究提供借鉴。     

MAGE-3 DNA疫苗的构建及其免疫效果的观察

通过RT PCR方法扩增MAGE 3cDNA ,以pcDNA3 1+为载体 ,构建重组表达质粒pcDNA3 1 MAGE 3。重组质粒用脂质体转染鼠B16细胞 ,经RT PCR、细胞免疫染色及免疫印迹法鉴定转化细胞中MAGE 3的表达。以 10 0 μg质粒剂量肌肉注射接种小鼠 ,间隔 10天 ,共 3次 ,以空载体和PBS为对照。结果 ,重组质粒免疫的小鼠 ,其脾淋巴细胞对MAGE 3阳性靶细胞的杀伤活性为 51 0 8± 7 41% ,与空载体组 (8 44± 1 89% )及PBS组 (5 76± 1 75% )相比 ,差异有显著性 (P <0 0 1) ,而对MAGE 3阴性靶细胞的杀伤活性分别为 8 2 1± 1 65% ,7 68± 1 56%和 5 13±1 42 % ,其差异无显著性 ;MAGE 3DNA疫苗组免疫血清 1∶15稀释时能检测到抗MAGE 3抗体 ,脾细胞培养上清中Th1类细胞因子IFN γ、IL 2水平明显升高 ,外周血中CD4+、CD8+T细胞也提高 ,小鼠肿瘤的生长速度明显减慢 ,与对照组相比 ,差异显著 (P <0 0 1)。说明MAGE 3重组质粒免疫小鼠可以诱导小鼠产生特异性的体液和细胞免疫应答     

小鼠Uncv基因克隆及真核表达

目的 克隆小鼠的Uncv基因并在真核细胞表达.方法 采用RT-PCR方法扩增小鼠皮肤组织中Uncv基因编码区,以真核表达质粒pcDNA 3.1-Flag为载体,构建Uncv真核表达质粒,将重组载体转染Hela细胞并用Western blot法检测基因表达.结果 构建Uncv基因真核表达载体pcDNA 3.1-Flag/Unev,重组质粒在Hela细胞中有效表达约95×103的融合蛋白.结论 成功构建真核表达载体pcDNA 3.1-Flag/Uncv,并且在真核细胞中有效表达,为研究Uncv基因生物学功能奠定基础.     

grp78真核表达载体构建及其稳定转染HeLa细胞系建立

目的构建人grp78基因真核表达载体,并建立稳定高表达grp78的人宫颈癌HeLa细胞系。方法用RT—PCR方法从人宫颈癌HeLa细胞中扩增grp78基因编码区,将PCR产物克隆到pcDNA3．1（＋）真核表达载体,构建重组质粒pcDNA3．1（＋）／grp78并测序鉴定。用构建成功的pcDNA3．1（＋）／grp78真核表达载体转染入人宫颈癌HeLa细胞,经G418筛选获得grp78稳定高表达的HeLa细胞系,并用RT—PCR及Western印迹方法鉴定。结果成功构建pcDNA3．1（＋）／grp78真核表达载体,筛选获得稳定高表达人grp78的HeLa细胞系。结论grp78真核表达载体的成功构建和稳定高表达grpTS的HeLa细胞系的建立为进一步研究grp78的功能奠定了基础。     

Differences in interleukin-12 and -15 production by dendritic cells at the early stage of Listeria monocytogenes infection between BALB/c and C57 BL/6 mice

The mechanisms responsible for the resistance of C57BL/6 mice and for the susceptibility of BALB/c mice to infection with Listeria monocytogenes were studied by comparing early IL-12 and IL-15 production by dendritic cells (DC) after infection with L. monocytogenes. Splenic DC expressing CD11b(low) and CD11c(+) obtained from C57BL/6 mice at 3 and 6 h after L. monocytogenes infection expressed higher levels of IL-12 p40 mRNA and IL-12 p40 protein than did those from BALB/c mice. Concurrently, a larger amount of IFN-gamma was produced by the splenic T cells from C57BL/6 mice in response to immobilized anti-TCRalphabeta mAb than by those from BALB/c mice, while the splenic T cells from BALB/c mice produced a higher level of IL-4 upon TCR alphabeta stimulation than did those of C57BL/6 mice. IL-15 mRNA and intracellular IL-15 protein were detected more abundantly in the DC from C57BL/6 mice than in those from BALB/c mice on day 3 after infection. CD3(+) IL2Rbeta (+) cells in the spleen were increased in C57BL/6 mice but not in BALB/c mice at the early stage after infection. Furthermore, IL-12Rbeta2 gene expression was up-regulated in T cells from C57BL/6 mice but not in those from BALB/c mice at the early stage after listerial infection. These results suggest that the difference in early production of IL-12 and IL-15 by DC may at least partly underlie the difference in susceptibility to L. monocytogenes between C57BL/6 and BALB/c mice.     

Transgene number-dependent, gene expression rate-independent rejection of Dd -, Kd-, or Dd Kd -transgened mouse skin or tumor cells from C57BL/6 Db Kb mice

Recipient cells migrating into the transplantation site of an allograft recognize histocompatibility antigens on the grafts and are cytotoxic against the grafts. Although the alloreactive immune response is predominantly directed at the major histocompatibility complex (major histocompatibility complex [MHC]; H-2 in mice) class I molecules, the basic mechanisms of allograft rejection (e.g., ligand-receptor interaction) remain unclear, because of the polymorphism and complexity of the MHC. To examine the role of MHC class I molecules in allograft rejection, D(d) , K(d) or D(d) K(d) -transgenic skin or tumor cells we established on a C57BL/6 (D(b) K(b) ) background and transplanted into C57BL/6 mice. Skin grafts from allogeneic (i.e., BALB/c, B10.D2, and BDF1) strains of mice were rejected from C57BL/6 mice on days 12-14 after grafting, whereas isografts were tolerated by these mice. Unexpectedly, skin grafts from D(d) -, K(d) -, and D(d) K(d) -transgenic C57BL/6 mice were rejected on days 12-14 in a transgene expression rate-independent manner from 9/19 (47%), 20/39 (51%), and 12/17 (71%) of C57BL/6 mice, respectively. Similarly, intradermally transplanted allogeneic (i.e., Meth A), but not syngeneic (i.e., EL-4), tumor cells were rejected from C57BL/6 mice; the growth of D(d) - or K(d) -transfected EL-4 cells was delayed by 10-13 days; and 4/10 (40%) of D(d) K(d) -transfected tumor cells were rejected from C57BL/6 mice. These results indicate that D(d) and K(d) genes are equivalent as allogeneic MHC class I genes and that C57BL/6 (D(b) K(b) ) mice reject D(d) -, K(d) -, or D(d) K(d) -transgened skin or tumor cells in a transgene number-dependent, gene expression rate-independent manner.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

Comparative study on high fat diet-induced 4-hydroxy-2E-nonenal adducts in the hippocampal CA1 region of C57BL/6N and C3H/HeN mice

In the present study, we investigated the influences of a high fat diet (HD) fed for 12 weeks, on lipid peroxidation and antioxidant enzyme using 4-hydroxy-2E-nonenal (HNE)-modified proteins (HNE-mp) and Cu,Zn-superoxide dismutase (SOD1) in the hippocampal CA1 region (CA1) in C57BL/6N and C3H/HeN mice. Body weights and body weight gains were significantly higher in HD fed C57BL/6N mice than in low fat diet (LD) fed C57BL/6N and LD or HD fed C3H/HeN mice. In the HD fed C57BL/6N and C3H/HeN mice, HNE-mp immunoreactivity and protein levels were much higher than in the LD fed C57BL/6N or C3H/HeN mice. In particular, HNE-mp immunoreactivity and protein levels in HD fed C57BL/6N mice was higher than that in the HD fed C3H/HeN mice. SOD1 immunoreaction was detected in the non-pyramidal cells of C57BL/6N mice, while in the C3H/HeN mice SOD1 immunoreaction was observed in CA1 pyramidal cells. The SOD1 immunoreactivity in the LD fed C57BL/6N and C3H/HeN mice was slightly, but not significantly decreased compared to that in the HD fed C57BL/6N and C3H/HeN mice, respectively. In addition, ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia in the HD fed C57BL/6N showed hypertrophy of cytoplasm, which is the characteristics of activated microglia. These results suggest that HD fed C57BL/6N mice are more susceptible to lipid peroxidation in the CA1 than in LD fed C57BL/6N and LD or HD fed C3H/HeN mice without any differences of SOD1 expression. In Koo Hwang and Il Yong Kim have contributed equally to this article.     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。