梅毒螺旋体膜蛋白DNA疫苗的优化及免疫策略的初步研究
发布时间：2018-08-26 16:14
【摘要】：梅毒螺旋体(Treponema pallidum, Tp)是人类性传播疾病(sexually transmitted disease, STD)梅毒的病原体。梅毒在STD中的致死性仅次于艾滋病,其不仅可严重损害成人人体的多个器官引起全身性损害,还可由母体经胎盘垂直传播给胎儿,引起胎儿全身性器官组织系统感染,导致早产、流产、死胎、畸胎或胎传梅毒,严重影响了出生人口质量。此外,梅毒与艾滋病的传播途径相同,还可大大增加感染、传播成人艾滋病和胎传艾滋病的危险性。尽管Tp的临床耐药株极其罕见,但近年来,梅毒发病率却居高不下,我国的梅毒感染率和发病率也呈直线上升趋势,近几年一直位于STD的前三位。因此,如何有效控制和预防梅毒,已成为全世界普遍关注的公共卫生问题,而制备出具有完全保护作用的梅毒疫苗则是其中的关键。 目前,国内外对Tp疫苗成分的研究主要集中于Tp重组外膜蛋白。Tp外膜蛋白既在Tp毒力中具有重要作用,也是宿主保护性免疫的主要靶标。由于Gpd和Tp92外膜蛋白序列高度保守且与其它致病性密螺旋体有高度的同源性,以及其较强的免疫原性和保护性,均被认为是研究Tp疫苗的最佳候选蛋白之一。本研究是在我们首次完成Tp单基因DNA疫苗筛选构建及免疫活性初步研究工作的基础上,将具有显著免疫佐剂效应的IL-2基因插入已真核表达重组体pcDNA3.1(+)/Gpd中,融合构建多价DNA疫苗,用壳聚糖(CS)纳米颗粒包被后导入新西兰兔体内,利用CS纳米颗粒高效的基因转导能力及IL-2的佐剂效应诱导动物细胞表达免疫原性更强的融合蛋白,强化免疫应答；同时采用核酸疫苗初免、含CpG序列的寡脱氧核苷酸(CpGODN)-Tp膜蛋白黏膜免疫加强的疫苗接种策略,以期全面激发机体免疫系统,尤其黏膜免疫,建立高效抗Tp感染的动物模型,为研制人用的高效疫苗预防梅毒打下基础。 目的： 1.在筛选Tp外膜蛋白基因Gpd和Tp92的单基因核酸疫苗在新西兰兔体内免疫活性的基础上,进一步构建Tp Gpd-IL-2基因融合壳聚糖纳米核酸疫苗,证实该类疫苗能否有效转染动物细胞,表达融合蛋白,诱导机体产生特异性免疫应答；证实细胞因子佐剂hIL-2和CS纳米颗粒能否有效辅佐疫苗强化免疫应答。 2.构建CpG ODN-Tp Gpd-IL-2重组膜蛋白黏膜疫苗；在上述基础上,应用核酸疫苗肌注初免-CpG ODN-蛋白黏膜疫苗加强免疫的接种策略,验证该策略能否全面激发机体免疫应答,特别是黏膜免疫应答。 3.建立Tp感染的新西兰兔动物模型,确切评价核酸疫苗肌注初免-CpG ODN-蛋白黏膜疫苗加强免疫的接种策略的免疫保护效果,为最终建立能完全有效抗Tp感染的动物模型,研制人用的高效疫苗打下良好基础。 方法： 1. Tp Nichols的传代与基因组提取：将低温冻存兔睾丸室温解冻后剪碎置于无菌生理盐水,让睾丸内保存的Tp Nichols株复苏、释放,再接种活兔睾丸传代,恢复毒力及活性后用于Tp感染实验及TpDNA基因组提取。 2.真核与原核表达重组体的构建：PCR钓取目的基因TpGpd、Tp92和hIL-2,与真核载体融合构建pcDNA3.1(+)/Gpd、 pcDNA3.1(+)/IL-2及pcDNA3.1(+)/Gpd-IL-2多价真核表达重组体；将目的基因IL-2与已成功构建的原核表达重组体(pET28a/Gpd)融合构建多价原核表达重组体(pET28a/Gpd-IL-2);测序鉴定。 3.真核与原核表达重组体的表达与鉴定：免疫印迹法鉴定pcD/TpGpd、pcD/Tp92、pcD/IL-2及pcD/Gpd-IL-2真核表达重组体在体外哺乳动物细胞内的表达；pET28a/TpGpd-IL-2原核表达重组体的蛋白表达、纯化、鉴定；将纯化的重组蛋白抗原免疫动物获取多抗。 4. pcD/TpGpd和pcD/Tp92单基因核酸疫苗免疫活性与保护性研究：将实验用兔随机分为pcD/Tp92疫苗组、pcD/TpGpd疫苗组、pcD空质粒对照组和PBS对照组4个小组；肌肉多点注射免疫,每2w免疫1次,共免疫3次。并于初次免疫后第8w每组随机取3只兔无菌分离脾细胞培养,MTT法检测兔脾淋巴细胞增殖水平,ELISA试剂盒检测脾细胞培养上清IL-2及IFN-γ诱导水平,在免疫及感染期间不同时间点分别采用ELISA检测免疫兔特异性抗体产生水平；第10w各实验组兔皮下接种TpNichols标准株进行皮肤感染实验,在感染后0-60d期间每隔3d观察记录感染部位皮损Tp阳性率及溃疡病灶发生率。 5.多基因疫苗的制备：制备CS纳米颗粒包裹核酸疫苗；制备CpG ODN-Gpd-IL-2蛋白黏膜疫苗。 6.壳聚糖纳米包裹核酸疫苗在新西兰兔中免疫活性及保护性研究：实验用兔随机分为12组,6个疫苗组(pcD/Gpd-IL-2+CS和pcD/Gpd-IL-2, pcD/Gpd+pcD/IL-2+CS和pcD/Gpd+pcD/IL-2, pcD/Gpd+CS和pcD/Gpd),6个对照组(pcD/IL-2+CS, pcD/IL-2, pcD+CS, pcD, CS和PBS组),肌肉多点注射免疫,每隔2w加强免疫一次共3次,并于初次免疫后第8w每组随机取3只兔无菌分离脾细胞培养,MTT法检测兔脾淋巴细胞增殖水平,ELISA试剂盒检测脾细胞培养上清IL-2及IFN-γ诱导水平,在免疫及感染期间不同时间点分别采用ELISA检测免疫兔特异性TpGpd抗体产生水平；第10w各实验组兔皮下接种TpNichols标准株进行皮肤感染实验,在感染后0-60d期间每隔3d观察记录感染部位皮损Tp阳性率及溃疡病灶发生率。比较、综合分析各类疫苗诱导的免疫应答水平及免疫保护效果。 7.核酸疫苗初免—蛋白黏膜疫苗加强免疫的接种策略评价：采用核酸疫苗肌注初免-CpGODN-蛋白黏膜疫苗加强免疫的疫苗接种策略,检测新西兰兔体内细胞免疫和体液免疫应答水平,评价免疫活性。对比评估核酸疫苗肌注初免—蛋白黏膜免疫加强的疫苗接种策略的抗Tp感染免疫保护效果。 结果： 1.成功复苏、传代Tp Nichols标准株并维持其良好的感染活性,可用于疫苗免疫后感染实验；成功制备Tp基因组DNA用于下一步研究。 2.构建的真核重组质粒pcD/Gpd-IL-2、pcD/Gpd、pcD/IL-2和pcD/Tp92经酶切和测序鉴定证实插入片段为目的基因,测序结果与Genbank上登录序列完全一致,均能在HeLa细胞内有效表达靶蛋白；构建的原核重组质粒pET28a/Gpd-IL-2能在Rosetta表达菌有效表达一个相对分子量(Mr)约60KD的融合蛋白。 3.pcD/TpGpd和pcD/Tp92单基因疫苗均能在免疫兔体内诱生比对照组(pcDNA3.1(+),PBS对照组)更高的抗TpGpd或Tp92特异性IgG抗体水平(P0.001),刺激更高的IL-2、IFN-γ细胞因子分泌(P0.001)及更强的脾细胞增殖分化(P0.001)。 4.pcD/Gpd-IL-2疫苗组和pcD/Gpd+pcD/IL-2联合疫苗组均能在兔体内诱生比pcD/Gpd单基因疫苗组更高的TpGpd特异性抗体水平(P0.05),刺激更高的IL-2、IFN-γ细胞因子分泌(P0.05)及更强的脾细胞增殖分化(P0.05)。 5.CS纳米颗粒对pcD/Gpd+pcD/IL-2联合疫苗组、pcD/Gpd-IL-2疫苗组及pcD/Gpd单基因疫苗的包裹未能显著促进相应疫苗组在兔体内诱生更高的的特异性抗体水平(P0.05),未能刺激更高的IL-2、IFN-γ细胞因子分泌(P0.05)及更强的脾细胞增殖分化(P0.05)。 6.Tp的兔皮肤感染导致感染期内各对照组特异性抗体水平及脾细胞增殖分化较之感染前有着显著升高(P0.05),对各疫苗组有一定促进作用；IL-2基因佐剂对各Tp Gpd DNA疫苗在感染期兔体内特异性抗体水平及脾细胞增殖分化的长久促进及维持有显著作用,而CS纳米颗粒对各Tp Gpd DNA疫苗的包裹未能显著提高在感染期阶段兔体内特异性抗体水平及脾细胞增殖分化。 7.各Tp Gpd DNA疫苗相比各对照组均能显著降低Tp感染部位皮损Tp阳性率及溃疡病灶形成率(P0.001),显示出较强的免疫保护效应；pcD/IL-2基因佐剂较之CS纳米颗粒更能增强pCD/TpGpd疫苗的免疫保护效果；两者联合使用可达到较之其它疫苗组更好的免疫效果。 8.在各实验组兔背部皮肤8个位点皮内Tp感染后0-60d同比观察比较,用CS+pCD/Gpd+pcD/IL-2联合疫苗和pCD/Gpd+pcD/IL-2联合疫苗,CS+pcD/Gpd-IL-2疫苗和pcD/Gpd-IL-2疫苗免疫兔后感染位点皮损红肿相对其它实验组直径最小,溃疡形成率最少,也最早愈合(42-45d),各组间差异不显著；PcD/Gpd和CS+pcD/Gpd疫苗组兔后感染位点皮损皮损红肿直径同比中等大小,两组间差异不显著；各对照组(CS+pcD/IL-2,pcD/IL-2,CS+pcD, pcD, CS+PBS,PBS对照组)感染位点皮损红肿直径最大,溃疡病灶形成数最多,也最晚愈合(60d)。 9.采用pcD/Gpd-IL-2疫苗肌注初免,CpGODN+Gpd-IL-2蛋白鼻饲加强免疫的接种策略既能刺激较强的体液和细胞免疫效应,与pcD/Gpd-IL-2疫苗肌注免疫组无显著差异,还能刺激较高的黏膜免疫效应,导致最低Tp感染部位皮损Tp阳性率(0%)及溃疡病灶形成率(3.33%)从而达到更有效地保护作用。 结论： 1.Tp92和TpGpd单基因DNA疫苗组经肌肉注射免疫新西兰兔均可诱生较强的体液免疫和细胞免疫应答,产生较好的保护作用。 2.IL-2基因与Tp Gpd抗原基因无论是融合表达还是非融合表达均能显著增强TpGpd单基因疫苗的免疫效应和免疫保护作用。 3.CS纳米颗粒包裹Tp Gpd DNA疫苗对疫苗的体液及细胞免疫效应及抗Tp皮肤感染有一定促进作用,但效果不显著。而CS纳米颗粒包裹加上IL-2基因佐剂的联合应用则能刺激pcD/Gpd疫苗产生更强的免疫效应及更好的抗Tp皮肤感染效果。 4.采用pcD/Gpd-IL-2疫苗肌注初免,CpGODN+Gpd-IL-2蛋白鼻饲加强免疫的接种策略,既能刺激机体产生较强的体液和细胞免疫效应,还能刺激较强的黏膜免疫效应,激发更有效的免疫保护作用。
[Abstract]:Treponema pallidum (Tp) is the pathogen of human sexually transmitted disease (STD). The lethality of syphilis in STD is second only to AIDS, which can not only seriously damage many organs of the adult human body and cause systemic damage, but also can be transmitted vertically from the mother to the fetus through the placenta, causing the whole fetus. Syphilis, like AIDS, can also greatly increase the risk of infection, transmission of adult AIDS and fetal transmission of AIDS. Although Tp clinical resistant strains are extremely rare, syphilis has developed in recent years. However, the prevalence rate of syphilis is still high, and the infection rate and incidence of syphilis in China are also on the rise. In recent years, syphilis has been in the top three of STD.
At present, the research on the components of Tp vaccine mainly focuses on the recombinant outer membrane protein of Tp. The outer membrane protein of Tp plays an important role in the virulence of Tp and is also the main target of host protective immunity. This study is based on the preliminary study on the screening and construction of a single gene DNA vaccine for Tp and its immunological activity. The IL-2 gene with significant immunoadjuvant effect was inserted into the eukaryotic expression recombinant pcDNA3.1 (+) / Gpd and fused to construct a multivalent DN. A vaccine, coated with chitosan (CS) nanoparticles, was introduced into New Zealand rabbits. The highly efficient gene transduction ability of CS nanoparticles and the adjuvant effect of IL-2 were used to induce the expression of more immunogenic fusion protein and enhance the immune response of animal cells. The aim of this study is to stimulate the immune system, especially the mucosal immunity, and to establish an animal model of highly effective anti-Tp infection, so as to lay a foundation for the development of a highly effective vaccine for the prevention of syphilis.
Objective:
1. On the basis of screening the monogenic nucleic acid vaccine of Tp outer membrane protein gene Gpd and Tp92 in New Zealand rabbits, the Tp Gpd-IL-2 gene fused chitosan nano-nucleic acid vaccine was further constructed to confirm whether the vaccine can effectively transfect animal cells, express fusion proteins and induce specific immune responses. Factor adjuvant hIL-2 and CS nanoparticles can effectively assist vaccines to enhance immune response.
2. To construct CpG ODN-Tp Gpd-IL-2 recombinant membrane protein mucosal vaccine; on the basis of the above, the strategy of reinforcing immunity by intramuscular injection of nucleic acid vaccine with primary immunization-CpG ODN-protein mucosal vaccine was applied to verify whether the strategy can fully stimulate the immune response of the body, especially the mucosal immune response.
3. Establishing the animal model of Tp infection in New Zealand rabbits and evaluating the immune protection effect of the strategy of enhancing immunity by intramuscular injection of nucleic acid vaccine with primary immunization-CpG ODN-protein mucosal vaccine.
Method:
1. The subculture and genomic extraction of Tp Nichols: The cryopreserved rabbit testis was thawed at room temperature and then cut into sterile saline. The Tp Nichols strain was resuscitated, released and inoculated into the living rabbit testis for passage. After recovery of virulence and activity, it was used in the experiment of Tp infection and the extraction of TpDNA genome.
2. Construction of eukaryotic and prokaryotic expression recombinants: TpGpd, Tp92 and hIL-2 were harvested by PCR and fused with eukaryotic vectors to construct pcDNA3.1 (+) / Gpd, pcDNA3.1 (+) / IL-2 and pcDNA3.1 (+) / Gpd-IL-2 multivalent eukaryotic expression recombinants; the target gene IL-2 was fused with the successfully constructed prokaryotic expression recombinant (pET28a / Gpd) to construct a multivalent prokaryotic expression recombinant (pET28 The recombinant plasmid (pET28a/Gpd-IL-2) was identified by sequencing.
3. Expression and identification of eukaryotic and prokaryotic expression recombinants: Western blotting was used to identify the expression of pcD/TpGpd, pcD/Tp92, pcD/IL-2 and pcD/Gpd-IL-2 eukaryotic expression recombinants in mammalian cells in vitro; pET28a/TpGpd-IL-2 prokaryotic expression recombinant protein was expressed, purified and identified; purified recombinant protein antigen was obtained from immunized animals. Polyclonal resistance.
4. Immune activity and protective effect of pcD/TpGpd and pcD/Tp92 single gene nucleic acid vaccine: The experimental rabbits were randomly divided into pcD/Tp92 vaccine group, pcD/TpGpd vaccine group, pcD empty plasmid control group and PBS control group; the rabbits were immunized by multi-point intramuscular injection, once every two weeks, three times in total, and randomly selected three rabbits in each group at the 8th week after the first immunization. Splenocyte culture was separated and MTT assay was used to detect the proliferation level of rabbit splenic lymphocytes, ELISA kit was used to detect the IL-2 and IFN-gamma induction level of splenic cell culture supernatant, and ELISA was used to detect the specific antibody production level of immunized rabbits at different time points during immunization and infection. The positive rate of Tp and the incidence of ulcer lesions were observed every 3 days from 0 to 60 days after infection.
5. Preparation of polygenic vaccine: preparation of CS nanoparticles encapsulated nucleic acid vaccine; preparation of CpG ODN-Gpd-IL-2 protein mucosal vaccine.
6. Immune activity and protective effects of chitosan nanoencapsulated nucleic acid vaccine in New Zealand rabbits: The experimental rabbits were randomrandomly divided into 12 groups, 6 vaccine groups (pcD/Gpd-IL-2+CSand pcD/Gpd-IL-2+CSand pcD/Gpd-IL-2, pcD/Gppcd+pcD/GpcD+pcD/IL-2+CSand pcD/Gppcd+Gpcd+pcd+pcd+pcD/IL-2+CSand pcD/Gpcd+pcd+pcd+pcd+pcd+pcd+pcd+pcd+pcd+pcd+pcD/IL multipoint Three rabbits were randomly selected from each group at the 8th week after the first immunization. MTT assay was used to detect the proliferation of splenic lymphocytes. ELISA kit was used to detect the levels of IL-2 and IFN-gamma in the culture supernatant of splenic cells. ELISA was used to detect the levels of IL-2 and IFN-gamma at different time points during the immunization and infection. The level of specific TpGpd antibody production in infected rabbits was measured. Subcutaneous inoculation with TpNichols standard strain was performed on rabbits in each experimental group on the 10th week. The positive rate of Tp and the incidence of ulcer lesions were observed and recorded every 3 days during the period of 0-60 days after infection.
7. Evaluation of inoculation strategy of primary immunization of nucleic acid vaccine-protein mucosal vaccine: The cellular immune and humoral immune responses of New Zealand rabbits were detected by using the strategy of primary immunization-CpGODN-protein mucosal vaccine intramuscular injection, and the immune activity was evaluated. The protective effect of vaccination against Tp infection is enhanced by membrane immunization strategy.
Result:
1. Successful resuscitation, passage of Tp Nichols standard strain and maintenance of its good infective activity can be used in post-vaccination infection experiments; Tp genomic DNA was successfully prepared for further research.
2. The constructed eukaryotic recombinant plasmids pcD/Gpd-IL-2, pcD/Gpd, pcD/IL-2 and pcD/Tp92 were confirmed to be the target genes by enzyme digestion and sequencing. The sequencing results were consistent with the Genbank login sequence and could effectively express the target protein in HeLa cells. A fusion protein with a relative molecular weight (Mr) of about 60KD.
3. Both pcD/TpGpd and pcD/Tp92 monogenic vaccines could induce higher levels of anti-TpGpd or Tp92 specific IgG antibodies (P 0.001), stimulate higher levels of IL-2, IFN-gamma cytokine secretion (P 0.001) and splenocyte proliferation and differentiation (P 0.001) in immunized rabbits than those in the control group (p cDNA 3.1 (+), PBS control group).
4. Both pcD/Gpd-IL-2 vaccine group and pcD/Gpd+pcD/IL-2 combined vaccine group could induce higher levels of TpGpd-specific antibodies (P 0.05), stimulate higher levels of IL-2, IFN-gamma cytokine secretion (P 0.05) and splenocyte proliferation and differentiation (P 0.05) in rabbits than pcD/Gpd monogenic vaccine group.
5. The encapsulation of CS nanoparticles on pcD/Gpd+pcD/IL-2 vaccine group, pcD/Gpd-IL-2 vaccine group and pcD/Gpd monogenic vaccine group did not significantly promote the production of higher specific antibodies in rabbits (P 0.05), but failed to stimulate higher IL-2, IFN-gamma cytokine secretion (P 0.05) and splenocyte proliferation and differentiation (P 0.05).
6. The levels of specific antibodies and the proliferation and differentiation of splenocytes in the control groups during the infection period were significantly higher than those before infection (P 0.05), and IL-2 gene adjuvant could promote the proliferation and differentiation of splenocytes in the infected rabbits. CS nanoparticles did not significantly increase the levels of specific antibodies and splenocyte proliferation and differentiation in rabbits during infection.
7. Compared with the control group, the Tp-Gpd DNA vaccines could significantly reduce the Tp positive rate and ulcer lesion formation rate (P 0.001), showing a strong immune protective effect; pcD/IL-2 gene adjuvant could enhance the immune protective effect of pCD/TpGpd vaccine more than CS nanoparticles; Better immune effect.
8. Compared with the control group, the rabbits immunized with CS+pCD/Gpd+pcD/IL-2 combined vaccine, pCD/Gpd+pcD/IL-2 combined vaccine, CS+pcD/Gpd-IL-2 vaccine and pcD/Gpd-IL-2 combined vaccine had the smallest diameter, the least ulcer formation rate and the earliest healing rate at the infected site (compared with the other experimental groups) 0-60 days after intradermal Tp infection at the 8 sites in the back skin of each experimental group. 42-45 days, there was no significant difference among the groups; PcD/Gpd and CS+pcD/Gpd vaccines had the largest redness and swelling diameter at the site of infection, the largest number of ulcer lesions and the latest healing of the lesions in the control group (CS+pcD/IL-2, pcD/IL-2, CS+pcD, pcD, CS+PBS, PBS control group). (60d).
9. Inoculation strategy of intramuscular immunization with pcD/Gpd-IL-2 vaccine and intranasal feeding of CpGODN+Gpd-IL-2 protein could stimulate strong humoral and cellular immune effects, which was not significantly different from that of intramuscular injection of pcD/Gpd-IL-2 vaccine. It could also stimulate higher mucosal immune effects, leading to the lowest Tp positive rate (0%) and ulcer lesions. (3.33%) to achieve more effective protection.
Conclusion:
1. Both Tp92 and TpGpd monogenic DNA vaccines can induce stronger humoral and cellular immune responses in New Zealand rabbits after intramuscular immunization, and produce better protective effects.
2. Both fusion and non-fusion expression of IL-2 gene and TpGpd antigen gene can significantly enhance the immune effect and protective effect of TpGpd vaccine.
3. CS nanoparticles encapsulated Tp-Gpd DNA vaccine can promote the humoral and cellular immunity of the vaccine and anti-Tp skin infection, but the effect is not significant. The combination of CS nanoparticles encapsulated with IL-2 gene adjuvant can stimulate pcD/Gpd vaccine to produce stronger immune effect and better anti-Tp skin infection effect.
4. The inoculation strategy of intramuscular immunization with pcD/Gpd-IL-2 vaccine and nasal feeding with CpGODN+Gpd-IL-2 protein can stimulate the body to produce strong humoral and cellular immune effects, but also stimulate strong mucosal immune effects, and stimulate more effective immune protection.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
本文编号：2205453
[Abstract]:Treponema pallidum (Tp) is the pathogen of human sexually transmitted disease (STD). The lethality of syphilis in STD is second only to AIDS, which can not only seriously damage many organs of the adult human body and cause systemic damage, but also can be transmitted vertically from the mother to the fetus through the placenta, causing the whole fetus. Syphilis, like AIDS, can also greatly increase the risk of infection, transmission of adult AIDS and fetal transmission of AIDS. Although Tp clinical resistant strains are extremely rare, syphilis has developed in recent years. However, the prevalence rate of syphilis is still high, and the infection rate and incidence of syphilis in China are also on the rise. In recent years, syphilis has been in the top three of STD.
At present, the research on the components of Tp vaccine mainly focuses on the recombinant outer membrane protein of Tp. The outer membrane protein of Tp plays an important role in the virulence of Tp and is also the main target of host protective immunity. This study is based on the preliminary study on the screening and construction of a single gene DNA vaccine for Tp and its immunological activity. The IL-2 gene with significant immunoadjuvant effect was inserted into the eukaryotic expression recombinant pcDNA3.1 (+) / Gpd and fused to construct a multivalent DN. A vaccine, coated with chitosan (CS) nanoparticles, was introduced into New Zealand rabbits. The highly efficient gene transduction ability of CS nanoparticles and the adjuvant effect of IL-2 were used to induce the expression of more immunogenic fusion protein and enhance the immune response of animal cells. The aim of this study is to stimulate the immune system, especially the mucosal immunity, and to establish an animal model of highly effective anti-Tp infection, so as to lay a foundation for the development of a highly effective vaccine for the prevention of syphilis.
Objective:
1. On the basis of screening the monogenic nucleic acid vaccine of Tp outer membrane protein gene Gpd and Tp92 in New Zealand rabbits, the Tp Gpd-IL-2 gene fused chitosan nano-nucleic acid vaccine was further constructed to confirm whether the vaccine can effectively transfect animal cells, express fusion proteins and induce specific immune responses. Factor adjuvant hIL-2 and CS nanoparticles can effectively assist vaccines to enhance immune response.
2. To construct CpG ODN-Tp Gpd-IL-2 recombinant membrane protein mucosal vaccine; on the basis of the above, the strategy of reinforcing immunity by intramuscular injection of nucleic acid vaccine with primary immunization-CpG ODN-protein mucosal vaccine was applied to verify whether the strategy can fully stimulate the immune response of the body, especially the mucosal immune response.
3. Establishing the animal model of Tp infection in New Zealand rabbits and evaluating the immune protection effect of the strategy of enhancing immunity by intramuscular injection of nucleic acid vaccine with primary immunization-CpG ODN-protein mucosal vaccine.
Method:
1. The subculture and genomic extraction of Tp Nichols: The cryopreserved rabbit testis was thawed at room temperature and then cut into sterile saline. The Tp Nichols strain was resuscitated, released and inoculated into the living rabbit testis for passage. After recovery of virulence and activity, it was used in the experiment of Tp infection and the extraction of TpDNA genome.
2. Construction of eukaryotic and prokaryotic expression recombinants: TpGpd, Tp92 and hIL-2 were harvested by PCR and fused with eukaryotic vectors to construct pcDNA3.1 (+) / Gpd, pcDNA3.1 (+) / IL-2 and pcDNA3.1 (+) / Gpd-IL-2 multivalent eukaryotic expression recombinants; the target gene IL-2 was fused with the successfully constructed prokaryotic expression recombinant (pET28a / Gpd) to construct a multivalent prokaryotic expression recombinant (pET28 The recombinant plasmid (pET28a/Gpd-IL-2) was identified by sequencing.
3. Expression and identification of eukaryotic and prokaryotic expression recombinants: Western blotting was used to identify the expression of pcD/TpGpd, pcD/Tp92, pcD/IL-2 and pcD/Gpd-IL-2 eukaryotic expression recombinants in mammalian cells in vitro; pET28a/TpGpd-IL-2 prokaryotic expression recombinant protein was expressed, purified and identified; purified recombinant protein antigen was obtained from immunized animals. Polyclonal resistance.
4. Immune activity and protective effect of pcD/TpGpd and pcD/Tp92 single gene nucleic acid vaccine: The experimental rabbits were randomly divided into pcD/Tp92 vaccine group, pcD/TpGpd vaccine group, pcD empty plasmid control group and PBS control group; the rabbits were immunized by multi-point intramuscular injection, once every two weeks, three times in total, and randomly selected three rabbits in each group at the 8th week after the first immunization. Splenocyte culture was separated and MTT assay was used to detect the proliferation level of rabbit splenic lymphocytes, ELISA kit was used to detect the IL-2 and IFN-gamma induction level of splenic cell culture supernatant, and ELISA was used to detect the specific antibody production level of immunized rabbits at different time points during immunization and infection. The positive rate of Tp and the incidence of ulcer lesions were observed every 3 days from 0 to 60 days after infection.
5. Preparation of polygenic vaccine: preparation of CS nanoparticles encapsulated nucleic acid vaccine; preparation of CpG ODN-Gpd-IL-2 protein mucosal vaccine.
6. Immune activity and protective effects of chitosan nanoencapsulated nucleic acid vaccine in New Zealand rabbits: The experimental rabbits were randomrandomly divided into 12 groups, 6 vaccine groups (pcD/Gpd-IL-2+CSand pcD/Gpd-IL-2+CSand pcD/Gpd-IL-2, pcD/Gppcd+pcD/GpcD+pcD/IL-2+CSand pcD/Gppcd+Gpcd+pcd+pcd+pcD/IL-2+CSand pcD/Gpcd+pcd+pcd+pcd+pcd+pcd+pcd+pcd+pcd+pcd+pcD/IL multipoint Three rabbits were randomly selected from each group at the 8th week after the first immunization. MTT assay was used to detect the proliferation of splenic lymphocytes. ELISA kit was used to detect the levels of IL-2 and IFN-gamma in the culture supernatant of splenic cells. ELISA was used to detect the levels of IL-2 and IFN-gamma at different time points during the immunization and infection. The level of specific TpGpd antibody production in infected rabbits was measured. Subcutaneous inoculation with TpNichols standard strain was performed on rabbits in each experimental group on the 10th week. The positive rate of Tp and the incidence of ulcer lesions were observed and recorded every 3 days during the period of 0-60 days after infection.
7. Evaluation of inoculation strategy of primary immunization of nucleic acid vaccine-protein mucosal vaccine: The cellular immune and humoral immune responses of New Zealand rabbits were detected by using the strategy of primary immunization-CpGODN-protein mucosal vaccine intramuscular injection, and the immune activity was evaluated. The protective effect of vaccination against Tp infection is enhanced by membrane immunization strategy.
Result:
1. Successful resuscitation, passage of Tp Nichols standard strain and maintenance of its good infective activity can be used in post-vaccination infection experiments; Tp genomic DNA was successfully prepared for further research.
2. The constructed eukaryotic recombinant plasmids pcD/Gpd-IL-2, pcD/Gpd, pcD/IL-2 and pcD/Tp92 were confirmed to be the target genes by enzyme digestion and sequencing. The sequencing results were consistent with the Genbank login sequence and could effectively express the target protein in HeLa cells. A fusion protein with a relative molecular weight (Mr) of about 60KD.
3. Both pcD/TpGpd and pcD/Tp92 monogenic vaccines could induce higher levels of anti-TpGpd or Tp92 specific IgG antibodies (P 0.001), stimulate higher levels of IL-2, IFN-gamma cytokine secretion (P 0.001) and splenocyte proliferation and differentiation (P 0.001) in immunized rabbits than those in the control group (p cDNA 3.1 (+), PBS control group).
4. Both pcD/Gpd-IL-2 vaccine group and pcD/Gpd+pcD/IL-2 combined vaccine group could induce higher levels of TpGpd-specific antibodies (P 0.05), stimulate higher levels of IL-2, IFN-gamma cytokine secretion (P 0.05) and splenocyte proliferation and differentiation (P 0.05) in rabbits than pcD/Gpd monogenic vaccine group.
5. The encapsulation of CS nanoparticles on pcD/Gpd+pcD/IL-2 vaccine group, pcD/Gpd-IL-2 vaccine group and pcD/Gpd monogenic vaccine group did not significantly promote the production of higher specific antibodies in rabbits (P 0.05), but failed to stimulate higher IL-2, IFN-gamma cytokine secretion (P 0.05) and splenocyte proliferation and differentiation (P 0.05).
6. The levels of specific antibodies and the proliferation and differentiation of splenocytes in the control groups during the infection period were significantly higher than those before infection (P 0.05), and IL-2 gene adjuvant could promote the proliferation and differentiation of splenocytes in the infected rabbits. CS nanoparticles did not significantly increase the levels of specific antibodies and splenocyte proliferation and differentiation in rabbits during infection.
7. Compared with the control group, the Tp-Gpd DNA vaccines could significantly reduce the Tp positive rate and ulcer lesion formation rate (P 0.001), showing a strong immune protective effect; pcD/IL-2 gene adjuvant could enhance the immune protective effect of pCD/TpGpd vaccine more than CS nanoparticles; Better immune effect.
8. Compared with the control group, the rabbits immunized with CS+pCD/Gpd+pcD/IL-2 combined vaccine, pCD/Gpd+pcD/IL-2 combined vaccine, CS+pcD/Gpd-IL-2 vaccine and pcD/Gpd-IL-2 combined vaccine had the smallest diameter, the least ulcer formation rate and the earliest healing rate at the infected site (compared with the other experimental groups) 0-60 days after intradermal Tp infection at the 8 sites in the back skin of each experimental group. 42-45 days, there was no significant difference among the groups; PcD/Gpd and CS+pcD/Gpd vaccines had the largest redness and swelling diameter at the site of infection, the largest number of ulcer lesions and the latest healing of the lesions in the control group (CS+pcD/IL-2, pcD/IL-2, CS+pcD, pcD, CS+PBS, PBS control group). (60d).
9. Inoculation strategy of intramuscular immunization with pcD/Gpd-IL-2 vaccine and intranasal feeding of CpGODN+Gpd-IL-2 protein could stimulate strong humoral and cellular immune effects, which was not significantly different from that of intramuscular injection of pcD/Gpd-IL-2 vaccine. It could also stimulate higher mucosal immune effects, leading to the lowest Tp positive rate (0%) and ulcer lesions. (3.33%) to achieve more effective protection.
Conclusion:
1. Both Tp92 and TpGpd monogenic DNA vaccines can induce stronger humoral and cellular immune responses in New Zealand rabbits after intramuscular immunization, and produce better protective effects.
2. Both fusion and non-fusion expression of IL-2 gene and TpGpd antigen gene can significantly enhance the immune effect and protective effect of TpGpd vaccine.
3. CS nanoparticles encapsulated Tp-Gpd DNA vaccine can promote the humoral and cellular immunity of the vaccine and anti-Tp skin infection, but the effect is not significant. The combination of CS nanoparticles encapsulated with IL-2 gene adjuvant can stimulate pcD/Gpd vaccine to produce stronger immune effect and better anti-Tp skin infection effect.
4. The inoculation strategy of intramuscular immunization with pcD/Gpd-IL-2 vaccine and nasal feeding with CpGODN+Gpd-IL-2 protein can stimulate the body to produce strong humoral and cellular immune effects, but also stimulate strong mucosal immune effects, and stimulate more effective immune protection.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
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