IL-18与HSV-2gD模拟抗原表位DNA疫苗的免疫效果观察

目的:研究串联重组核酸疫苗pcDNA3.1-IL-18-HSVP6和pcDNA3.1-IL-18-HSVNP6对机体的免疫效果。NP6为与HSV-2gD模拟抗原P6最相似的天然抗原表位序列(Accession Number:E00394)。方法:将构建的真核表达质粒pcDNA3.1-IL-18、pcDNA3.1-IL-18-HSVP6和pcDNA3.1-IL-18-HSVNP6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;末次免疫后一月,处死小鼠,无菌分离脾脏,MTT法测定脾淋巴细胞增殖率。结果:重组核酸疫苗pcDNA3.1-IL-18-HSVP6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:重组核酸疫苗pcDNA3.1-IL-18-HSVP6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。     

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。     

HSV-2gD、HBsAg和IL18多表位基因疫苗的构建方法

目的研究多表位串联重组核酸疫苗中IL18与HBsAg(S)抗原、P6(NP6)抗原在质粒中所处位置的差异对抗原表达是否有影响。方法分别将IL18连接在融合蛋白P6-S(NP6-S)的氨基端和羧基端,即构建4种质粒:pc-IL18-P6-S、pc-IL18-NP6-S、pc-S-P6-IL18和pc-S-NP6-IL18。采用DNAstar软件分析4种质粒表达的融合蛋白的抗原性。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 blotting检测S、P6(NP6)抗原的表达情况,以确定最佳的连接方式。结果应用DNAstar软件分析可知IL18在融合蛋白的氨基端的抗原性高于在羧基端。间接免疫荧光检测结果发现IL18位于融合蛋白羧基端的质粒表达的抗原性优于氨基端,即:质粒pc-S-P6-IL18和pc-S-NP6-IL18的S、P6(NP6)抗原的表达情况均优于质粒pc-IL18-P6-S和pc-IL18-NP6-S,故选用质粒pc-S-P6-IL18和pc-S-NP6-IL18作为优势质粒。结论成功构建了4种质粒,并确定了优势质粒,此项技术将为新一代多基因核酸疫苗的研制提供实验依据,从而为构建更加有效的多价DNA疫苗奠定了良好的基础。     

HSV-1 VP22和hIL-18增强HBV DNA微球疫苗诱导的小鼠体液免疫应答

为了研究HSV-1VP22和hIL-18作为分子佐剂对HBVDNA微球疫苗诱导小鼠体液免疫应答的影响,HSV病毒经Vero细胞培养增殖后,提取病毒DNA,PCR扩增VP22编码基因。从人PBMC中提取总RNA,RT-PCR扩增hIL-18。VP22、hIL-18按不同需要插入pcDNA3.1-S,构建成3种质粒:pcDNA3.1-VP22-S、pcDNA3.1-S-IL-18、pcDNA3.1-VP22-S-IL-18。制备DNA壳聚糖微球疫苗,鼻腔免疫小鼠,同时裸质粒DNA肌注小鼠,ELISA检测小鼠血清IgG、粪便sIgA水平。结果,与pcDNA3.1-S相比,pcDNA3.1-VP22-S、pcDNA3.1-S-IL-18、pcDNA3.1-VP22-S-IL-18均能引起小鼠血清IgG、粪便sIgA水平升高。在鼻腔免疫实验中,pcDNA3.1-VP22-S、pcDNA3.1-S-IL-18、pcDNA3.1-VP22-S-IL-18免疫组的血清IgG水平均在第8周达到最高,并且与pcDNA3.1-S相比,抗体水平有差异(P<0.05)。研究结果表明,VP22和hIL-18能够增强乙肝DNA疫苗的体液免疫应答;制备的DNA微球疫苗经鼻腔免疫后,能够诱导黏膜免疫应答。     

幽门螺杆菌Lpp20核酸疫苗的构建及其免疫活性的初步研究

目的 构建幽门螺杆菌脂蛋白Lpp20基因的真核表达载体pcDNA3.1(+)/Lpp20,并在HeLa细胞中进行表达.通过肌肉注射免疫C57BL/6小鼠,观察其诱导小鼠产生的体液免疫和细胞免疫应答水平.方法 用PCR法扩增Lpp20全基因,再将Lpp20基因克隆至pcDNA3.1(+)真核细胞表达载体构建pcDNA3.1(+)/Lpp20重组体,观察其在HeLa细胞中的表达.将核酸疫苗PcDNA3.1(+)/Lpp20、对照空质粒pcDNA3.1(+)及PBS分组通过肌肉注射免疫6周龄C57BL/6小鼠.隔2周免疫一次,共免疫4次.间接ELISA法测定小鼠血清中抗Lpp20 IgG抗体水平,双抗体夹心ELISA法检测脾淋巴细胞培养上清中IFN-γ水平,MTT比色法检测脾淋巴细胞增殖反应.通过PCR法检测小鼠肌细胞中Lpp20基因的存在.结果 小鼠接种pcDNA3.1(+)/Lpp20核酸疫苗后能产生特异性IgG抗体,6周后ELISA测定血清抗体A450值为0.74,效价为1:1024.核酸疫苗免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ含量明显升高[(410.36±56.23)ps/ml],与空质粒组[(25.26±10.85)pg/ml]之间差异有统计学意义(P<0.01).脾淋巴细胞增殖反应测定,核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数(2.37±0.22)明显高于空质粒组(1.53±0.47)和PBS组(1.20±0.13),P<0.01.PCR检测Lpp20基因可在小鼠肌细胞中存在.结论 成功构建了pcDNA3.1(+)/Lpp20核酸疫苗,且其在小鼠体内可诱导较强的特异性体液免疫和细胞免疫应答.为进一步研究该疫苗的免疫保护作用提供实验依据.     

沙眼衣原体主要外膜蛋白核酸疫苗诱导小鼠产生特异性细胞和体液免疫应答

目的构建沙眼衣原体主要外膜蛋白核酸疫苗,并观察其诱导小鼠产生的体液免疫和细胞免疫。方法将核酸疫苗(pcDNA3.1MOMP)或对照空质粒(pcDNA3.1)注射于4～6周龄小鼠后腿股四头肌,每次剂量为100mg。间隔2周加强免疫2次。末次免疫后,ELISA法测定脾淋巴细胞培养上清液中IFNγ及小鼠血清中抗MOMP水平;MTT法测定脾淋巴细胞特异性增殖反应。结果小鼠接种核酸疫苗后,能产生特异性抗体,第3次免疫后抗体最高滴度达1∶1024,培养上清液中IFNγ达(532.0±45.4)pg/mL;实验组小鼠脾淋巴细胞刺激指数为3.94±0.25,其抗原特异性反应明显高于对照组。结论沙眼衣原体主要外膜蛋白核酸疫苗能刺激机体产生特异的细胞免疫和体液免疫。     

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。     

人β防御素-2与前列腺癌特异性膜抗原嵌合蛋白真核表达质粒构建及其诱导的小鼠特异性免疫应答

探索人β防御素2 (h BD- 2 )和前列腺特异性膜抗原(PSMA)共表达重组核酸疫苗针对前列腺癌的免疫治疗。研究以pc DNA3.1为载体,构建重组质粒pc DNA3.1/PSMA和pc DNA3.1/h BD- 2 - PSMA,通过RT- PCR和免疫组化检测其表达。免疫小鼠后,进行血清中抗体检测,CD4 、CD8 T淋巴细胞数目测定及CTL 特异性杀伤作用检测。结果显示构建的质粒转染COS- 7细胞后能表达目的基因,免疫小鼠后能在体内持久表达,可以诱导产生特异性抗体,能有效的刺激T细胞增生,诱导特异性CTL 反应。当以h BD- 2作为免疫佐剂时,CTL 活性更强。本研究成功的构建了含PSMA的表达质粒,免疫小鼠可以诱导出有效的体液和细胞免疫,为前列腺癌的免疫治疗奠定了一定的实验基础     

狂犬病毒糖蛋白基因重组腺病毒与同一抗原核酸疫苗的联合免疫研究

目的 研究联合运用狂犬病毒糖蛋白基因重组腺病毒与同一抗原核酸疫苗对小鼠的免疫效果。方法 构建狂犬病毒糖蛋白（GP）的真核表达质粒pcDNA3.1/CVS-N2c GP作为核酸疫苗，瞬时转染COS－7细胞，间接免疫荧光试验检测pcDNA3.1/CVS-N2c GP的表达；以基因枪方法进行初次免疫接种和首次加强免疫，以表达同一抗原的复制缺陷型重组腺病毒通过鼻腔接种进行第二次加强免疫；ELISA试验检测血清狂犬病毒特异性IgG抗体，快速荧光灶抑制试验（RFFTT）检测狂犬病毒中和抗体。结果 间接免疫荧光试验表明pcDNA3.1/CVS-N2c GP能有效地表达GP于转染的细胞膜上，ELISA试验表明小鼠接受基因核核酸免疫后，仅诱导产生低水平的特异性抗体，而且重组腺病毒进行加强免疫后，特异性抗体水平显著提高。结论 联合运用狂犬病毒糖蛋白基因重组腺病毒与同种抗原核酸疫苗可克服它们单独使用时各自的缺点，能有效地诱导小鼠产生抗狂犬病毒特异免疫。     

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。     

The role of neutralizing antibody and T-helper subtypes in protection and pathogenesis of vaccinated mice following ocular HSV-1 challenge.

In order to determine the possible correlation of specific immune responses with protection against mortality and ocular disease following ocular herpes simplex virus type 1 (HSV-1) challenge, BALB/c mice were vaccinated with different doses and regimens of baculovirus-expressed gD. Neutralizing antibody, virus titres in the eyes, corneal scarring, and survival were measured. In addition, infiltration into the cornea of CD4+ T cells and cells containing the lymphokines interleukin-2 (IL-2), IL-4, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were monitored on days 3, 7, 10, 14 and 21 post-challenge by immunocytochemistry. The vaccination regimens used induced varying degrees of immune responses and protection upon ocular challenge with HSV-1. Our results suggest that neutralizing antibody was the most important immune response in protecting mice against lethal ocular challenge and corneal scarring. TNF-alpha and IL-2 were not crucial in terms of survival and corneal scarring, since gD1 (one vaccination with 1 microg of gD) and gD0.1 (one vaccination with 0.1 microg of gD), both of which provided high levels of protection, showed no TNF-alpha or IL-2 expression. However, TNF-alpha and IL-2 were crucial in terms of virus clearance from the eyes, since gD3 (three vaccinations with 1 microg of gD), which had less virus in their eyes, had high numbers of TNF-alpha and IL-2 infiltrates. Finally, mock-vaccinated mice were not protected from death and corneal disease following HSV-1 challenge. Eyes of mock-vaccinated mice had little or no TNF-alpha or IL-2 responses and the strongest IL-4 and IL-6 responses.     

壳聚糖介导增强幽门螺杆菌Lpp20 DNA疫苗黏膜免疫效果的研究

目的 探讨壳聚糖包裹DNA疫苗黏膜免疫效果.方法 采用复凝聚法制备载幽门螺杆菌脂蛋白 Lpp20基因的壳聚糖(CS)纳米粒(NPs),并对CS/DNA纳米粒的特质进行研究;用载基因壳聚糖纳米粒黏膜免疫(滴鼻和口服)小鼠,检测免疫小鼠的细胞和体液免疫水平.结果 裸质粒pcDNA3.1(+)/Lpp20与CS/DNA NPs通过黏膜免疫均能诱导小鼠产生有效的免疫应答.CS/DNANPs滴鼻和口服免疫组诱导的抗体明显升高,与裸质粒组相比有明显差异(P＜0.05),同时CS/DNANPs滴鼻和口服免疫组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数及培养上清中IFN-γ和IL-4含量明显高于裸质粒组、壳聚糖组和PBS组,且滴鼻免疫组高于口服组.结论 壳聚糖纳米粒能增强pcDNA3.1(+)/Lpp20核酸疫苗的黏膜免疫(滴鼻和口服免疫)效果;载Lpp20基因壳聚糖纳米粒滴鼻免疫比口服免疫能诱导更强的细胞和体液免疫应答.

Abstract：

Objective To investigate the immune response of mucosal immunization of new chitosan(CS) nanoparticles coating DNA vaccine. Methods The chitosan nanoparticles containing plasmid DNA encoding H. pylori lipoprotein Lpp20 gene were prepared using a complex coacervation method and then its speciality were analyzed. We then administered the naked plasmid DNA and chitosan-DNA nanoparticles to 6-week-old female BALB/c mice by intranasal or oral mucosal routes to observe the humoral and cellular immune responses. Results Naked plasmid pcDNA3.1 ( + )/Lpp20 and chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles both induced effective immune response in mice through mucosal vaccination. Specific IgG and sIgA antibodies of chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles groups were higher than that of naked pcDNA3.1 ( + )/Lpp20 group. The concentration of cytokines IFN-γ and IL-4 in cultural supernatant of T lymphocytes from chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles immunized mice increased greatly than that of control groups. After stimulated by corresponding antigen, the stimulation index of intranasal or oral delivery of chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles group was significantly higher than that of pcDNA3.1( + )/Lpp20 group, CS group and PBS control group. Moreover, systemic and mucosal immune responses in mice induced by intranasal immunization were stronger than that of oral immunization. Conclusion Chitosan nanoparticles enhanced the immune response of pcDNA3.1 ( + )/Lpp20 DNA vaccine by intranasal or oral administration in BALB/c mice. Compared to oral administration, intranasal delivery of chitosan-pcDNA3.1 ( + )/Lpp20 DNA nanoparticles could induce stronger cellular and humoral immune responses in BALB/c mice.     

重组质粒pcDNA3.1IL-2/Fc对HBV preS2S DNA疫苗免疫作用的影响

目的 探讨IL-2/Fc融合表达后对HBVpreS2S基因疫苗诱导免疫反应的佐剂效应.方法 采用HBV preS2S DNA疫苗作为基础免疫,重组质粒pcDNA3.1IL-2/Fc作为佐剂加强免疫BALB/c小鼠,采用0、2、4周的方案接种,检测各次接种后抗体水平.初次免疫后7周,测定免疫脾细胞的杀伤活性、增殖活性和细胞因子的分泌水平.结果 pcDNA3.1IL-2/Fc作为佐剂在HBV preS2S注射3 d后免疫组小鼠抗体滴度、免疫脾细胞的杀伤活性和增殖活性、TH1型细胞因子的分泌水平,均比各对照组明显增强.结论 IL-2/Fc是有效的HBV preS2S DNA疫苗佐剂之一.     

Coexpression of interleukin-2 enhances the immunization effect of a DNA vaccine expressing herpes simplex 1 glycoprotein D

In this study, DNA vaccines consisting of vector IRES-gD expressing Herpes simplex virus 1 (HSV-1) glycoprotein D (gD) and vector IRES-gD-IL-2 coexpressing HSV-1 gD and interleukin-2 (IL-2), respectively, were constructed. After intramuscular inoculation, both vaccines induced in BALB/c mice antibodies as assayed by ELISA and virus neutralization. However, IRES-gD-IL-2 elicited significantly higher levels of IgG (ELISA) and neutralizing antibodies than IRES-gD. Isotyping of sera from mice injected with IRES-gD-IL-2 revealed predominantly IgG2a antibodies. IRES-gD-IL-2 also elicited a higher delayed-type sensitivity (DTH) reaction. However, there was no difference in the protection against lethal challenge with HSV-1 between the two vaccines (P>0.05). The results suggest that the vaccination with IRES-gD-IL-2 can efficiently enhance the immune response of mice to HSV-1, particularly through increased cellular immunity.     

人偏肺病毒DNA疫苗的构建和小鼠免疫反应的研究

目的 构建人偏肺病毒(hMPV)DNA疫苗,小鼠免疫后评价其细胞和体液免疫水平.方法 利用PCR方法,从hMPV的cDNA中扩增融合蛋白FATM(缺失跨膜区)基因和基质蛋白M基因,构建DNA疫苗pcDNA3.1His-FATM和pcDNA3.1His-M,瞬时转染后用Western Blot和间接免疫荧光方法检测F、M蛋白表达.疫苗肌内注射免疫小鼠,ELISA和ELISPOT方法分别检测血清IgG抗体和小鼠脾细胞CTL水平.结果 Western Blot和间接免疫荧光(IFA)方法证明构建的疫苗可表达FATM和M蛋白.peDNA3.1His-FATM单独免疫小鼠,血清抗体滴度为1:44;与pcDNA3.1His-M联合免疫后,血清抗体滴度为1:64.ELISPOT检测证明,联合免疫组小鼠脾细胞产生IFN-γ的效应CD8+T细胞数为42±8.9,高于单独免疫组32±7.4的水平.结论 DNA疫苗peDNA3.1His-F△TM可以诱导产生特异性的体液和细胞免疫,与pcDNA3.1His-M联合免疫,可以提高免疫水平.