The protective immunity produced in infected C57BL/6 mice of a DNA vaccine encoding Schistosoma japonicum Chinese strain triose-phosphate isomerase

The development of a SjCTPI DNA vaccine for Schistosoma japonicum and the detection of the immune responses to and the protective efficacy of immunization were performed and challenged in C57BL/6 mice. According to the gene sequence of SjCTPI and murine IL-12, three pairs of primers were designed. The full length cDNA encoding SjCTPI and P35, P40 amplified from pUC19-SjCTPI and murine IL-12 by PCR were subcloned into an eukaryotic expression vector (pcDNA3.1). Forty-five female C57BL/6 mice were divided into three groups; each mouse of the control group was injected with 100 pg of pcDNA3.1 by i.m. route; the TPI group was injected with 100 microg of pcDNA3. 1-SjCTPI; the TPI+IL- 12 group was injected with 100 microg of pcDNA3.1-SjCTPI and 100 pg of mixture of pcDNA3.1-P35 and pcDNA3.1-P40. Each mouse was immunized at weeks 1 and 5 and challenged with 45 cercariae of Schistosoma japonicum Chinese strain at week 9. The mice were killed and perfused 45 days after challenge; the numbers of recovered worms and hepatic eggs were counted. The expression of SjCTPI in muscle tissue was determined by an immunohistochemical method. Culture of spleen cells showed the production of IL-2, IL-4, IL-10 and IFN-gamma with the stimulation of specific antigen before and after challenge. Sera were collected from each group before immunization, before challenge and two weeks post challenge; ELISA and Western-blot tests were performed for detection of anti-rTPI antibodies. The antigen of SjCTPI was expressed in the membrane and plasma of the muscle cells of C57BL/6 mice. The obvious rising of IL-2 in TPI group and TPI+IL-12 group before and after challenge was seen. The anti-rTPI antibody detection with Western-blot showed that ten serum samples from the control group were negative; nine of ten serum samples from the TPI group were weakly positive, eight of ten from the TPI+IL-12 group were weakly positive. The worm and egg reduction rates of TPI group and TPI+IL- 12 group were 27.9% and 13.7%, 31.9% and 18.6% respectively in comparison with the pcDNA group. pcDNA3.1-TPI DNA vaccine could confer partial protection against a subsequent challenge of Schistosoma japonicum in C57BL/6 mice and might therefore be a potential DNA vaccine.     

体内电穿孔增强日本血吸虫核酸疫苗免疫保护作用的研究

目的探讨体内电穿孔技术增强日本血吸虫核酸疫苗的免疫保护效果。方法分别大量制备质粒pcDNA3．1-SjC23、pcDNA3．1-SjCTPI、pcDNA3．1-（CDR3）6和重组蛋白SjC23-HD、SjCT-PI与NP30。上述3种质粒DNA以等量混合后即为鸡尾酒式DNA疫苗，3种蛋白以等量混合后即为鸡尾酒式蛋白疫苗。70只BALB／c小鼠随机分为A、B、C、D、E5组，每组14只。A组为自然感染组；B组（电脉冲空质粒对照组）每只小鼠分别在第0、3、6周经股四头肌注射100μl pcDNA3．1，每次注射时辅以体内电穿孔；C组（电脉冲空质粒＋混合蛋白对照组）空质粒免疫及体内电穿孔同B组，但于第9周每鼠经背部皮下多点注射100μl混合蛋白疫苗＋100μl福氏完全佐剂（FCA）；D组（电脉冲混合DNA组）每只小鼠分别在第0、3、6周经股四头肌注射100μl混合DNA疫苗，每次免疫时辅以体内电穿孔；E组（电脉冲混合DNA＋混合蛋白组）混合DNA免疫及体内电穿孔同D组，但于第9周每鼠经背部皮下多点注射100弘1混合蛋白疫苗＋100μl FCA。DNA免疫组末次免疫后4周，蛋白加强组末次免疫后2周，所有小鼠同时经腹部皮肤感染（40±1）条尾蚴。攻击感染后42d剖杀小鼠，计数成虫及肝脏虫卵数。首次免疫前2d及感染前2d分别经尾静脉采血，分离血清检测kG抗体水平、抗体亚类IgGl及IgG2a，并取小鼠脾脏制备单个脾细胞，检测细胞因子IL-2、IL-4、Ifn-y的水平。结果C、D组和E组的减虫率分别为18．09％、45．00％和57．09％，D组和E组的减虫率均显著高于C组（P均〈0．01），且E组的减虫率高于D组（P〈0．05）；C、D组和E组的减卵率分别为12．49％、50．88％和59．26％，D组和E组的减卵率均显著高于C组（P均〈0．01），且E组的减卵率高于D组（P〈0．05）。C、D、E3组小鼠血清都检测到特异性IgG抗体，抗体亚类IgG2a／IgGl比值分别为0．394、3．518、0．914。D     

Protective immunity induced with 23 kDa membrane protein dna vaccine of Schistosoma japonicum Chinese strain in infected C57BL/6 mice

A 23 kDa membrane protein DNA vaccine for Schistosoma japonicum Chinese strain was developed and tested for its protective efficacy and immune responses in infected C57BL/6 mice. The cDNA encoding SjC23 amplified from pUC19-SjC23 were subcloned into an eukaryotic expression vector (pcDNA3.1). Forty-eight female C57BL/6 mice were divided into three groups. Each mouse of group A (control group) was immunized intramuscularly (i.m.) with 100 microg of pcDNA3.1; of group B (SjC23 group) was immunized (i.m.) with 100 microg of pcDNA3.1-SjC23; of group C (SjC23+IL-12) was immunized (i.m.) with a mixture of 100 microg of pcDNA3.1-SjC23, 100 microg of pcDNA3.1-p35 and 100 microg of pcDNA-p40. These were followed by two boosts of the same DNA once every two weeks. All mice were challenged with 45 cercariae of Schistosoma japonicum Chinese strain at week 8, and were killed and perfused at week 14. The numbers of recovered worms and hepatic eggs were counted. The expression of SjC23 and p35, p40 in muscle tissue was determined by immunohistochemical method. By culture of spleen cells, the production of IL-2, IL-4, IL-10 and IFN-gamma with the stimulation of specific antigen of the recombinant hydrophilic domain of SjC23 (rSjC23-HD) was determined after the last immunization (before challenge). Sera were collected from each group before immunization and two weeks before and after challenge. Anti-SjC23 antibodies were tested by Western blot. The results showed that SjC23 and p35, p40 of mouse IL-12 were expressed on the membrane and in the plasma of the muscle cells of immunized C57BL/6 mice. A rise of IL-2 and IFN-gamma in the SjC23 group and SjC23+IL-12 group was observed; No changes were found in IL-4 and IL-10. Detection of anti-SjC23 antibody with Western blot showed that after the third immunization (before challenge) all the serum samples from the control group were negative; 8 of 10 sera from the SjC23 group and 9 of 10 sera from the SjC23+IL-12 group were positive. The worm reduction rates in the SjC23 group and SjC23+IL-12 group were 26.9% and 35.4% respectively; the liver eggs reduction rates were 22.2% and 28.4%, respectively in comparison to the control group. This indicates that the pcDNA3.1-SjC23 DNA vaccine can induce partial protection against Schistosoma japonicum infection in C57BL/6 mice.     

日本血吸虫TPI DNA疫苗基因密码子优化增强免疫保护作用的研究

目的　 研究日本血吸虫中国大陆株磷酸丙糖异构酶基因（TPI基因）密码子优化后的DNA疫苗增强免疫保护作用的效果。 方法　 60只BALB/c雌性小鼠随机均分为A（pcDNA3.1空质粒对照组）、B（pcDNA3.1-TPI组）、C （pcDNA3.1-TPI-mHSP70组）、D（pcDNA3.1-TPI.opt组）、E（pcDNA3.1-TPI.opt-mHSP70组）等5组。每鼠肌肉注射相应的纯化质粒DNA 100 μg,每隔3周免疫1次,共3次。末次免疫后4周,每鼠经腹部皮肤攻击感染日本血吸虫尾蚴（40±1）条,42 d后剖杀,计数成虫及肝脏虫卵数。首次免疫前2 d及感染前2 d经尾静脉采血,检测IgG及IgG₁、IgG_2a的水平。攻击感染前2 d取脾脏,制备单个脾细胞,用流式细胞仪检测白细胞介素2（IL-2）、IL-4、IL-5、γ干扰素（IFN-γ）及肿瘤坏死因子（TNF）的水平。 结果　 B、C、D、E组小鼠血清均检测到特异性IgG及IgG_2a与IgG₁抗体,IgG_2a/IgG₁的比值分别为1.73、2.06、2.44、3.09。D、E组的IL-2、IFN-γ、TNF含量较B、C组均有不同程度地升高。D、E组减虫率分别为36.03%、39.03%,减卵率分别为41.71%、46.85%,均显著高于B、C组（P<0.01）。 结论 TPI基因密码子优化后的DNA疫苗相对于未优化TPI DNA疫苗能诱导小鼠产生较高的免疫保护作用,且诱导宿主产生较强的,及以Th1为主的免疫应答。     

乙型肝炎表面抗原与粒细胞巨噬细胞集落刺激因子融合表达质粒免疫乙型肝炎病毒转基因小鼠的研究

目的 研究乙型肝炎表面抗原(HBsAg)和粒细胞巨噬细胞集落刺激因子(GM-CSF)的融合表达质粒免疫乙型肝炎病毒(HBV)转基因小鼠及其病毒清除作用。方法 将小鼠随机分为8组,分别注射以下质粒:A组pcDNA3.1-S 100μg;B组:pcDNA3.1-GM-CSF-S 100μg;C组:pcDNA3.1-S-GM-CSF 100μg;D组:pcDNA3.1-S 50μg pcDNA3.1-GM-CSF 50μg;E组:pcDNA3.1-GM-CSF 100μg;F组:重组酵母乙型肝炎疫苗1μg;G组:pcDNA3.1 100μg;H组:磷酸盐缓冲液(PBS)100μl。于质粒注射前4d肌肉注射0.25％bupivacaine溶液100μl,诱导骨骼肌细胞变性。以HBsAg和GM-CSF的融合乙型肝炎表达质粒免疫HBV转基因小鼠,检测HBV转基因小鼠血清HBsAg表达水平及肝细胞HBsAg的表达,检测脾细胞白细胞介素(IL)-2、IL-4及γ干扰素(IFN-γ)的分泌水平及淋巴细胞增殖情况,并进行肝组织学检查。结果 质粒加强免疫4周后血清HBsAg检测结果A组为28.28±15.32、B组为10.77±9.18、C组为44.16±8.30、D组为21.93±13.28、E组48.04±3.60、F组为38.3±11.66、G组为47.03±3.96、H组为51.46±4.70,F=11.262,P<0.01,其中B组血清HBsAg滴度显著低于其余各组。IL-2分泌水平(pg/ml)A组为25.5±7.5、B组为48.5±11.3、C组为4.0±2.5、D组为38.0±8.5、E组为3.7±2.1、F组为6.5±23.     

日本血吸虫调宁蛋白样蛋白P14基因DNA疫苗对小鼠免疫保护作用研究

摘 要：目的 研究日本血吸虫调宁蛋白样蛋白P14基因DNA疫苗对小鼠免疫保护作用。 方法 制备无内毒素DNA疫苗,用于免疫小鼠。将雌性BALB/c小鼠随机分为4组,每组10只。生理盐水组给予100 μl/鼠/次;空质粒组100 μg/鼠/次、pcDNA3.1(+)-SjP14组、pcDNA3.1(+)-SjP14 + pcDNA3.1(+)-SjGST组分别经肌肉给予100 μg/鼠/次;同上每2周免疫一次,共3次。末次免疫后2周,经腹部皮肤感染日本血吸虫尾蚴（30±1）条/鼠。尾蚴攻击6周后解剖小鼠,收集成虫和血清,计算减虫率并检测血清IgG1、IgG2a及总IgG;同时留取肝脏,部分消化后在显微镜下行虫卵计数,计算减卵率,部分肝脏用于组织病理学分析（HE染色法）,观察肝细胞变化及肉芽肿情况。结果 与NS对照组比较,pcDNA3.1(+)-Sj P14p核酸疫苗组减虫率和减卵率分别达到45.1%（P<0.05）和62.0%（P<0.001）;SjP14核酸疫苗与SjGST核酸疫苗联合免疫组减虫率和减卵率分别提高至56.3%（P<0.01）和73.9%（P<0.001）;SjGST和SjP14 组减虫率大于SjP14疫苗组,但两组之间差异无显著性（P＞0.05）; SjGST和SjP14组减卵率明显大于SjP14疫苗组, 两组之间差异有显著性（P<0.001）。SjGST和SjP14组与SjP14组血清IgG1、IgG2a及总IgG水平在免疫6周、12周后均较对照组显著提高（P<0.01）,但SjGST和SjP14组与SjP14之间差异无显著性（P＞0.05）。肝组织切片镜下显示,pcDNA3.1(+)-SjP14组肝脏病变较生理盐水组和空质粒组明显减轻,pcDNA3.1(+)-SjP14 + pcDNA3.1(+)-SjGST组肝脏损伤程度最轻,虫卵肉芽肿周围炎症反应轻,肉芽肿面积较小。结论 pcDNA3.1(+)-Sj P14核酸疫苗有一定程度的抗血吸虫感染作用,pcDNA3.1(+)-SjP14与pcDNA3.1(+)-SjGST疫苗联合免疫能增强小鼠对血吸虫感染的保护。     

Sjcb2 DNA疫苗在血吸虫感染小鼠中的保护性作用研究

目的 研究Sjcb2 DNA疫苗在日本血吸虫病小鼠模型中的保护性作用和机制,为血吸虫疫苗的研究提供有效的候选抗原分子。方法 构建pcDNA3.1(+)/Sjcb2 核酸疫苗,将6周龄雌性BALB/c小鼠随机分为pcDNA3.1(+)/Sjcb2 核酸疫苗组、pcDNA3.1(+)空质粒组及生理盐水组,每组35只,采用后腿股四头肌注射方法,每次免疫质粒DNA 100 μg,每2周免疫1次,共免疫3次,用日本血吸虫尾蚴攻击感染各组小鼠。PCR及免疫组化法检测Sjcb2基因在小鼠体内的稳定性及表达情况;MTT法检测小鼠脾淋巴细胞特异性增殖反应;ELISA法检测小鼠血清中Sjcb2抗体水平及攻击感染前后脾淋巴细胞培养上清中IFN-γ和IL-4的水平;计数小鼠荷成虫对数和肝脏荷虫卵数。结果 疫苗组小鼠均可在小鼠肌细胞中检测到Sjcb2基因及其抗原的表达;DNA疫苗组T细胞增殖显著增高(P <0.05);ELISA 结果显示疫苗组IFN-γ 水平显著增高(P <0.05),血吸虫尾蚴攻击感染后各组小鼠IL-4水平显著升高(P <0.05)。DNA疫苗组小鼠荷成虫对数及肝脏荷虫卵数与其它组比较显著性减少(P <0.05),其减虫率为36.32％,减卵率为60.61％。结论 Sjcb2 DNA疫苗接种小鼠后能在小鼠肌细胞中稳定存在和表达;Sjcb2可能通过提高IFN-γ 和降低IL-4水平调节Th1细胞亚群产生抗血吸虫感染的保护性作用。     

日本血吸虫中国大陆株23 kDa膜蛋白DNA疫苗诱导小鼠保护性免疫的研究

目的　研究日本血吸虫中国大陆株 2 3kDa膜蛋白 (SjC2 3)DNA疫苗诱导C5 7BL/6小鼠免疫保护作用。方法　将全长的SjC2 3基因克隆到真核表达载体pcDNA3.1,构建DNA疫苗 pcDNA3.1 SjC2 3。制备SjC2 3及IL 12的两个亚单位 p35、p4 0的DNA疫苗和对照 pcDNA3.1。 4 8只C5 7BL/6小鼠随机分为A、B、C 3组。A组小鼠肌注 10 0 μgpcDNA3.1;B组注射 10 0 μg pcDNA3.1 SjC2 3;C组肌注 pcDNA3.1 SjC2 3、pcDNA3.1 p35及pcDNA3.1 p4 0各 10 0 μg的混合物。每隔 2周各免疫 1次 ,共 3次。第 8周每鼠感染 4 5± 2条 /只尾蚴 ,4 5d后剖杀 ,计数成虫及肝内虫卵。采用免疫组化法检测SjC2 3及 p35、p4 0在小鼠局部组织内的表达 ;用脾细胞培养法检测经rSjC2 3 HD刺激后 ,攻击前、后小鼠脾细胞IL 2、IL 4、IL 10和IFN γ的水平。用Westernblotting检测血清中抗SjC2 3抗体。结果　SjC2 3以及p35、p4 0在免疫小鼠股四头肌细胞膜和细胞浆均获得表达。IL 2和IFN γ的水平攻击前、后在B组和C组均明显升高。Westernblotting检测抗SjC2 3抗体结果表明 ,免疫后两周 ,B组 8/10份血清为阳性 ,C组 9/10份血清阳性。B组和C组分别获得 2 6 .9%和 35 .4 %的减虫率 ,C组显著高于B组 (P <0 .0 5 ) ;减卵率分别为 2 2 .2 %和 2 8.4 %。结论　SjC     

日本血吸虫鸡尾酒式DNA疫苗与蛋白疫苗联合应用增强免疫保护作用的研究

目的 探讨日本血吸虫鸡尾酒式DNA疫苗与蛋白疫苗联合应用以增强免疫保护作用的效果。方法分别大量制备质粒DNA：pcDNA3．1-SjC23、pcDNA3．1-SjCTPI、pcDNA3．1-（CDR3）6和重组蛋白SjC23-HD、SjCTPI、NP30。pcDNA3．1-SjC23、pcDNA3．1-SjCTPI、pcDNA3．1-（CDR3）6等量混合后即为鸡尾酒式的混合DNA疫苗，重组蛋白SjC23-HD、SjCTPI、NP30等量混合后即为鸡尾酒式的混合蛋白疫苗。70只BALB／c小鼠随机分为A、B、C、D、E5组，每组14只。A组为自然感染组；B组（空质粒对照组）每只小鼠分别在第0、3、6周经股四头肌注射100μlpcDNA3．1；C组（空质粒＋混合蛋白对照组）每只小鼠分别在第0、3、6周经股四头肌注射100μlpcDNA3．1，第9周每鼠经背部皮下多点注射100μl混合蛋白疫苗＋100μl福氏完全佐剂（FCA）；D组（混合DNA组）每只小鼠分别在第0、3、6周经股四头肌注射100μl混合DNA疫苗；E组（混合DNA＋混合蛋白组）每只小鼠分别在第0、3、6周经股四头肌注射100μl混合DNA疫苗，第9周每鼠经背部皮下多点注射100μl混合蛋白疫苗＋100μlFCA。DNA免疫组末次免疫后4周，蛋白加强组末次免疫后2周，所有小鼠同时经腹部皮肤感染（40±1）条尾蚴。攻击感染后42d剖杀小鼠，计数成虫及肝脏虫卵数。首次免疫前2d及感染前2d分别经尾静脉采血，分离血清检测IgG抗体水平、抗体亚类IgG1及IgG2a，并取小鼠脾脏制备单个脾细胞，检测细胞因子IL-2、IL-4、IFN-γ的水平。结果C、D组和E组的减虫率分别为17．70％、32．88％和45．35％，D组和E组的减虫率均显著高于C组（P均〈0．01），且E组的减虫率显著高于D组（P〈0．01）；C、D组和E组的减卵率分别为9．39％、36．20％和48．54％，D组和E组的减卵率均显著高于C组（P均〈0．01），且E组的减虫率也显著高于D组（P〈0．05）。C、D、E3组小鼠血清都检测到特异性IgG抗体，?     

弓形虫主要表面抗原1核酸疫苗与蛋白疫苗联合免疫保护作用的研究

目的 研究刚地弓形虫RH株主要表面抗原1(P30)DNA疫苗诱导BALB/c小鼠的保护性免疫作用。方法 根据弓形虫P30基因的DNA序列设计一对引物,,将PCR扩增到的P30基因克隆到真核表达载体pcDNA.3.1中。大量制备pcDNA3. 1-P30和pcDNA3.1质粒DNA。将48只 BALB/c小鼠随机分成4组,每组1 2只,空质粒对照组(A组)第O、2、4周经小鼠股四头肌注射100 μg pcDNA3.1质粒DNA;重组P30抗原免疫组(B组)第0、2、4周每鼠经背部皮下多点注射50 μg rP30+福氏完全佐剂;P30 DNA疫苗免疫组(C组)第O、2、4周经小鼠股四头肌注射100 μg pcD- NA3. 1-P30质粒DNA;P30 DNA疫苗和重组P30抗原联合免疫组(D组)第O、2周经小鼠股四头肌洼射100 μg pcDNA3. 1-P30质粒DNA,第4周每鼠经背部皮下多点注射50 μg rP30+福氏完全佐剂。末次免疫4周后每鼠用100个弓形虫速殖子经腹腔感染,观察小鼠存活时间。结果 成功构建刚地弓形虫RH株P30DNA疫苗,动物保护性实验表明,虽然与对照组相比实验组小鼠的存活时间有一定的延长,但差异无显著性。结论 pcDNA3.1-P30 DNA疫苗具有弓形虫病候选DNA疫苗分子的潜力。     

HBV DNA vaccine with adjuvant cytokines induced specific immune responses against HBV infection

AIM: To seek for an effective method to improve the immuneresponses induced by DNA vaccine expressing HBV surfaceantigen (pCR3.1-S) in Balb/c mice (H-2d).METHODS: The pCR3.1-S plasmid and the eukaryoticexpression vectors expressing murine IL-2 (pDOR-IL-2) orIL-12 (pWRG3169) were injected into mice subcutaneously.The immune responses to pCR3.1-S and the adjuvant effectof the cytokines plasmid were studied. Meanwhile the effectof pCR3.1-S on anti-translated subcutaneous tumor of P815mastocytoma cells stably expressing HBsAg (P815-HBV-S)was also studied. Anti-HBs in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HBsAg specificcytotoxic T lymphocytes (CTLs) activity was measured by 51Crrelease assay. After three weeks of DNA immunization, thecells of P815-HBV-S were inoculated into mice subcutaneouslyand the tumor growth was measured every five days. Thesurvival rate and living periods of mice were also calculated.RESULTS: After 8 wk DNA immunization, the ,4 450 nmvalues of sera in mice immunized with pCR3.1, pCR3.1-Sand pCR3.1-S codeliveried with IL-2 or IL-12 plasmids were0.03±0.01, 1.24±0.10, 1.98±0.17 and 1.67±0.12respectively. Data in mice codeliveried pCR3.1-S with IL-2or IL-12 plasmids were significantly higher than that of miceinjected pCR3.1 or pCR3.1-S only. The HBsAg specific CTLactivities in mice coinjected with pCR3.1-S and IL-2 or IL-12 eukaryotic expression vectors were (61.9±7.1) % and(73.3±8.8) %, which were significantly higher than that ofmice injected with pCR3.1 (10.1±2.1) % or pCR3.1-S (50.5±6.4) %. The HBsAg specific CTL activities in mice injectedwith pCR3.1, pCR3.1-S, pCR3.1-S combined with IL-2 or IL-12 eukaryotic expression vectors decreased significantly to(3.2±0.8) %, (10.6±1.4) %, (13.6±1.3) % and (16.9±2.3)% respectively after the spleen cells were treated by anti-CD8+ monoclonal antibody, but presented no significantchange to anti-CD4+ monoclonal antibody or unrelated tomonoclonal antibody. The HBV-S DNA vaccine (pCR3.1-S)could evidently inhibit the tumor growth, prolong the survivalperiod of mice and improve the survival rate of mice andthese effects could be improved by IL-12 gene codeliveried.CONCLUSION: HBV DNA vaccine has a strong antigenicityin humoral and cellular immunities, which can be promotedby plasmid expressing IL-2 or IL-12. CD8+ cells executedthe CTL activities. DNA vaccine may be useful for bothprophylaxis and treatment of HBV infection.     

HBV DNA vaccine with adjuvant cytokines induced specific immune responses against HBV infection

AIM: To seek for an effective method to improve the immuneresponses induced by DNA vaccine expressing HBV surfaceantigen (pCR3.1-S) in Balb/c mice (H-2d).METHODS: The pCR3.1-S plasmid and the eukaryoticexpression vectors expressing murine IL-2 (pDOR-IL-2) orIL-12 (pWRG3169) were injected into mice subcutaneously.The immune responses to pCR3.1-S and the adjuvant effectof the cytokines plasmid were studied. Meanwhile the effectof pCR3.1-S on anti-translated subcutaneous tumor of P815mastocytoma cells stably expressing HBsAg (P815-HBV-S)was also studied. Anti-HBs in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HBsAg specificcytotoxic T lymphocytes (CTLs) activity was measured by 51Crrelease assay. After three weeks of DNA immunization, thecells of P815-HBV-S were inoculated into mice subcutaneouslyand the tumor growth was measured every five days. Thesurvival rate and living periods of mice were also calculated.RESULTS: After 8 wk DNA immunization, the ,4 450 nmvalues of sera in mice immunized with pCR3.1, pCR3.1-Sand pCR3.1-S codeliveried with IL-2 or IL-12 plasmids were0.03±0.01, 1.24±0.10, 1.98±0.17 and 1.67±0.12respectively. Data in mice codeliveried pCR3.1-S with IL-2or IL-12 plasmids were significantly higher than that of miceinjected pCR3.1 or pCR3.1-S only. The HBsAg specific CTLactivities in mice coinjected with pCR3.1-S and IL-2 or IL-12 eukaryotic expression vectors were (61.9±7.1) % and(73.3±8.8) %, which were significantly higher than that ofmice injected with pCR3.1 (10.1±2.1) % or pCR3.1-S (50.5±6.4) %. The HBsAg specific CTL activities in mice injectedwith pCR3.1, pCR3.1-S, pCR3.1-S combined with IL-2 or IL-12 eukaryotic expression vectors decreased significantly to(3.2±0.8) %, (10.6±1.4) %, (13.6±1.3) % and (16.9±2.3)% respectively after the spleen cells were treated by anti-CD8+ monoclonal antibody, but presented no significantchange to anti-CD4+ monoclonal antibody or unrelated tomonoclonal antibody. The HBV-S DNA vaccine (pCR3.1-S)could evidently inhibit the tumor growth, prolong the survivalperiod of mice and improve the survival rate of mice andthese effects could be improved by IL-12 gene codeliveried.CONCLUSION: HBV DNA vaccine has a strong antigenicityin humoral and cellular immunities, which can be promotedby plasmid expressing IL-2 or IL-12. CD8+ cells executedthe CTL activities. DNA vaccine may be useful for bothprophylaxis and treatment of HBV infection.     

日本血吸虫Sj23DNA疫苗的增效研究

目的 探讨缺失肿瘤转移有关基因ME491同源序列的日本血吸虫Mr23 000膜蛋白基因（Sj23DNA）突变体疫苗的免疫效果。 方法 将Sj23DNA中与人黑素瘤细胞膜上的ME491同源序列进行基因缺失突变，再将突变基因转染真核细胞人胚肾HEK293细胞，通过间接荧光抗体试验（IFAT）检测其表达情况。将pcDNA3-Sj23突变体经股四头肌注射免疫小鼠（100 μg/只），免疫后4周取注射部位肌肉作冰冻切片，IFAT检测目的基因的表达。40只BALB/c小鼠随机分成4组（每组10只），分别经股四头肌注射pcDNA3-Sj23突变体、pcDNA3-Sj23、空白质粒pcDNA3（均100 μg/只）和生理盐水（30 μl/只）免疫1次。免疫后4周用尾蚴攻击感染（40±2条/只）。第42天剖杀，肠系膜静脉灌注法及挤压法计数成虫，取肝脏计算每克肝组织虫卵数（EPG）。以减虫率和减卵率评价其免疫保护力。 结果 pcDNA3-Sj23突变体疫苗可在真核细胞HEK293中瞬时表达。pcDNA3-Sj23突变体在小鼠注射部位骨骼肌细胞上出现较强荧光，空白对照组未见特异性荧光。pcDNA3-Sj23突变体获得40.3%减虫率和42.8%减卵率，pcDNA3-Sj23获得33.1%减虫率和28.9%减卵率。两组差异均有统计学意义（P值均<0.05）。 结论 与pcDNA3-Sj23相比，缺失ME491同源序列的cDNA3-Sj23突变体疫苗能诱导BALB/c小鼠产生更好的免疫保护效果。     

免疫刺激序列增强日本血吸虫DNA疫苗的免疫保护作用

目的　探讨免疫刺激序列在日本血吸虫Mr 23 000膜蛋白 (SjC23)DNA疫苗诱导BALB/c小鼠抗血吸虫感染中的作用。　方法　将SjC23基因片段克隆到增加了免疫刺激序列的真核表达质粒 pcDNA3.1-CpG中,构建pcDNA3.1-SjC23/CpG。 40只雌性BALB/c小鼠随机分为 4组 ,① pcDNA3.1对照组 ;②pcDNA3.1-SjC23组 ;③ pcDNA3.1-CpG组 ;④ pcDNA3.1-SjC23/CpG组。每鼠经两侧股四头肌注射质粒DNA共100 μg ,隔 2周加强免疫 1次 ,共 3次。末次免疫后 4周经腹部皮肤感染日本血吸虫尾蚴 45条 /鼠 ,45d后计数成虫及肝脏虫卵数。首次免疫前和感染前 2d分别经尾静脉采血 ,检测IgG及IgG1、IgG2a。末次免疫后 3周取小鼠脾细胞 ,检测经伴刀豆球蛋白和SjC23重组蛋白刺激后小鼠白细胞介素 2 (IL-2 )、白细胞介素 4(IL-4)和γ干扰素 (IFN-γ)。用51Cr释放法检测经SjC23重组蛋白刺激后脾细胞对小鼠淋巴瘤细胞的杀伤作用。　结果　②组和④组减虫率分别为 2 8.1%和 3 5.1% ,减卵率分别为 2 1.6%和 2 6.5 %。④组减虫率显著高于②组 (P <0.0 5 )。这两组均检测到特异性IgG ,IgG2a/IgG1比值分别为 10.1和 12.2。脾细胞经伴刀豆球蛋白和SjC23重组蛋白刺激后的IL-2水平 ,②组较①组、④组较③组均有升高。②组脾细胞对靶细胞的杀伤活性为9.7%, ④组为40.0%。 结论 疫苗载体中增加免疫刺激序列，可提高SjC23 DNA 疫苗在BALB/c小鼠中诱导产生的免疫保护作用。     

磷酸铝佐剂对乙型肝炎DNA疫苗抗体应答的增强作用

目的 探讨磷酸铝佐剂对乙型肝炎DNA疫苗诱导体液免疫应答的增强作用。 方法 应用基因重组技术构建乙型肝炎表面抗原(HBsAg)真核表达质粒pcDNA 3.1-S,经酶切和测序鉴定无误后,作为乙型肝炎DNA疫苗,将不同浓度的磷酸铝悬液与之混合后免疫小鼠;用酶联免疫吸附法检测重组质粒在小鼠局部肌肉中HBsAg的表达和在血清中HBsAg的含量;并于免疫小鼠6周后检测小鼠血清抗-HBs水平。 结果 与单纯使用质粒pcDNA 3.1-S相比,将质粒pcDNA 3.1-S与磷酸铝悬液混合后免疫小鼠,重组质粒在小鼠局部肌肉中HBsAg表达差异无显著性;HBsAg在血清中的浓度均为阴性。将质粒pcDNA3.1-S与磷酸铝悬液混合后免疫小鼠,6周后检测小鼠血清抗-HBs,每1μl质粒中含1、10、50、100 μg磷酸铝组,小鼠血清抗-HBs抗体的P/N值分别为11.00±6.62、20.30±10.20、49.1 8±24.40和48.68±27.78,单纯使用质粒pcDNA3.1-S组P/N值为11.54±5.60。含1μg和10μg磷酸铝组,抗-HBs抗体P/N值与单纯用质粒pcDNA3.1-S组相比,差异无显著性;50μg和100μg磷酸铝组抗-HBs抗体P/N值高于单纯用质粒pcDNA3.1-S组,但50μg和100μg磷酸铝组二者差异无显著性。 结论 磷酸铝与质粒pcDNA3.1-S混合对pcDNA3.1-S在小鼠局部肌肉中HBsAg的表达无明显增强作用,但一定含量的磷酸铝能显     

白细胞介素-12在日本血吸虫脂肪酸结合蛋白诱导小鼠保护性免疫力中的佐剂作用

目的 研究白细胞介素-12（IL-12）对日本血吸虫脂肪酸结合蛋白（Sj14FABP）DNA疫苗的免疫增强效果。方法　 分别构建pVIVO2-Sj14FABP 和pVIVO2-IL12-Sj14FABP疫苗，鉴定构建成功后免疫小鼠。48只雄性BALB/c小鼠随机分为A、B、C和D等4组, 分别用0.9% NaCl（对照组）、pVIVO2、pVIVO2-Sj14FABP和pVIVO2-IL12-Sj14FABP肌注免疫1次（100 μg/只）。免疫后30 d各组小鼠感染40±2条尾蚴, 感染后45 d剖杀, 计数成虫及肝内虫卵；同时用ELISA法检测小鼠血清中IgG抗体水平，用双夹心ELISA法检测脾淋巴细胞经伴刀豆球蛋白A（ConA）和可溶性虫卵抗原（SEA）刺激后培养上清中白细胞介素-2（IL-2）、白细胞介素-4（IL-4）和干扰素-γ（IFN-γ）含量。 结果 经PCR和酶切鉴定，成功构建pVIVO2-Sj14FABP和pVIVO2-IL12-Sj14FABP重组质粒；C、D组免疫后分别获得24.1%和39.4%的减虫率、27.2%和32.8%的减卵率；细胞因子检测结果，D组经ConA和SEA诱生的IL-2和IFN-γ水平显著增高（P　<　0.01），而IL-4水平显著降低，差异有统计学意义（P　<　0.01）；免疫后30 　d小鼠血清IgG水平无明显升高，各实验组间差异也无统计学意义(P　>0.05)。 结论 Sj14FABP DNA疫苗可诱导BALB/c小鼠产生部分抗血吸虫感染保护作用, 细胞因子IL-12能够诱导机体免疫反应向Th1型优势分化，并增强血吸虫病DNA疫苗免疫保护性效果。     

重组Sj14-3-3,SjGST及mIL-12/聚乳酸聚乙醇酸共聚物(PLGA)缓释微球对小鼠免疫保护性的研究

目的研究聚乳酸聚乙醇酸共聚物(PLGA)微球给药系统在血吸虫病分子疫苗中的应用,并探讨rSj14-3-3,rSjGST及mIL-12作为疫苗的协同作用,及mIL-12刺激机体产生CTL和辅助性T细胞(Th)在抗血吸虫病中的作用。方法从pET28a/Sj14-3-3和GST重组质粒中诱导表达rSj14-3-3及rSjGST,过柱纯化。构建真核表达质粒pcDNA3.1(+)mIL-12,并共同包入PLGA缓释微球。对制备的微球进行体外释放,观察其释放速度。分组免疫BALB/c小鼠,进行尾蚴攻击感染实验。在攻击感染6w后,剖杀小鼠,计算各组的减虫率。结果各组的减虫率:单独rSj14-3-3组为27.6%,rSj14-3-3+PcDNA3.1(+)mIL-12组34.9%,rSj14-3-3+PcDNA3.1(+)mIL-12PLGA微球组37.5%,rSj14-3-3与rSjGST混合后+PcDNA3.1(+)mIL-12PLGA微球组38.8%;各组减卵率分别为(按以上组序)35.3%,49.1%,50.5%,和43.1%。结论PLGA微球给药系统可诱导并调节体液与细胞免疫从而增强了疫苗的抗感染,抗生殖作用。但rSj14-3-3和rSjGST抗原之间未表现出协同作用。mIL-12刺激机体产生CTL和辅助性T细胞(Th),在BALB/c鼠抗血吸虫攻击感染发挥作用,增强了疫苗保护作用。     

日本血吸虫体表蛋白Tetraspanin 2-A核酸疫苗的构建及其对小鼠的免疫试验

采用PCR法扩增日本血吸虫体表四跨膜家族蛋白2-A（SjTsp2-A）基因,构建重组质粒pcDNA3.1（+）/SjTsp2-A,将其转至大肠埃希菌DH5α制备DNA疫苗pcDNA3.1（+）/SjTsp2-A。24只BALB/c小鼠均分3组,每鼠于左股四头肌注射0.5 mg/ml盐酸布比卡因50 μl。次日,A组同法注射DNA疫苗pcDNA3.1（+）/SjTsp2-A,B组注射重组质粒pcDNA3.1（+）/SjGST,C组注射空质粒pcDNA3.1（+）。注射剂量均为100 μg/只。每隔2周注射1次,共3次,末次免疫后2周各组均经腹部皮下感染日本血吸虫尾蚴40±2条/鼠,45 d后剖杀,计数减虫率和减卵率。ELISA检测抗体效价,A组化分析股四头肌局部组织蛋白表达情况。结果A组的平均检虫数和每克肝组织虫卵数均显著低于B组和C组（P值均<0.05）,A组的减虫率和减卵率分别为44.4%和28.4%。A组血清抗体效价高达1 ∶ 25 600。A、B两组局部组织均有特异性蛋白表达。DNA候选疫苗pcDNA3.1（+）/SjTsp2-A能诱导小鼠产生一定的免疫保护作用。     

日本血吸虫组织蛋白酶B DNA疫苗与IL-4真核表达质粒联合免疫诱导小鼠保护性的研究

目的　观察日本血吸虫组织蛋白酶BDNA疫苗与IL 4真核表达质粒联合免疫小鼠的效果。　方法　将小鼠IL 4基因PCR扩增片段克隆入真核表达载体pcDNA3以构建重组表达质粒。小鼠分为 4组 ,每组 12只 ,实验组 (A)每鼠肌注组织蛋白酶BDNA疫苗和IL 4表达质粒各 10 0 μg ,同时设立组织蛋白酶BDNA疫苗对照组 (B)、IL 4表达质粒对照组 (C)和空载体对照组 (D) ,共免疫 3次。 2周后用免疫组化检测表达质粒在小鼠肌细胞的表达 ,3周后经皮肤攻击感染小鼠 40± 1条日本血吸虫尾蚴。计算减虫和减卵率 ,观察免疫保护性。　结果　重组IL 4质粒和组织蛋白酶BDNA疫苗均在小鼠肌细胞表达。用重组IL 4质粒和组织蛋白酶BDNA疫苗联合免疫诱导小鼠产生 43 .2 0 %的减虫率和 76.63 %的减卵率 ,与组织蛋白酶BDNA疫苗单独免疫比较差异均有显著性 (P <0 .0 0 1,P <0 .0 5 )。　结论　联合IL 4表达质粒免疫可能提高日本血吸虫组织蛋白酶BDNA疫苗的抗血吸虫保护性免疫。     

嗜肺军团菌flaA基因DNA疫苗的构建及其免疫保护性研究

目的构建flaA基因的DNA疫苗,观察其对嗜肺军团菌感染的免疫保护作用。方法用限制性核酸内切酶从重组质粒pET32a-flaA上切下flaA基因,亚克隆到真核表达载体pcDNA3.1(+),重组子经限制性酶切分析、PCR鉴定正确后,命名为pcDNA3.1-flaA。将18只BALB/c雌性小鼠随机分为3组,pcDNA3.1-flaA实验组肌肉接种pcDNA3.1-flaA质粒100μg,2w后加强免疫一次,2个对照组分别肌肉注射等体积的生理盐水和100μg空质粒,加强免疫后3w小鼠鼻腔滴注嗜肺军团菌Lp1菌液进行攻击感染,感染4w后,检查小鼠肺组织带菌量,观察肺组织病理形态学改变。结果成功构建了真核表达重组质粒pcDNA3.1-flaA,攻击感染实验表明,pcDNA3.1-flaA免疫组肺组织带菌量显著少于生理盐水对照组和空质粒对照组(P<0.05),且肺组织病理变化症状较pcDNA3.1对照组和生理盐水对照组的肺轻微。结论由flaA基因构建的嗜肺军团菌DNA疫苗能诱导小鼠产生保护性免疫。