發(fā)布時(shí)間：2018-08-26 16:14

【摘要】：梅毒螺旋體(Treponema pallidum, Tp)是人類性傳播疾病(sexually transmitted disease, STD)梅毒的病原體。梅毒在STD中的致死性僅次于艾滋病,其不僅可嚴(yán)重?fù)p害成人人體的多個(gè)器官引起全身性損害,還可由母體經(jīng)胎盤垂直傳播給胎兒,引起胎兒全身性器官組織系統(tǒng)感染,導(dǎo)致早產(chǎn)、流產(chǎn)、死胎、畸胎或胎傳梅毒,嚴(yán)重影響了出生人口質(zhì)量。此外,梅毒與艾滋病的傳播途徑相同,還可大大增加感染、傳播成人艾滋病和胎傳艾滋病的危險(xiǎn)性。盡管Tp的臨床耐藥株極其罕見,但近年來(lái),梅毒發(fā)病率卻居高不下,我國(guó)的梅毒感染率和發(fā)病率也呈直線上升趨勢(shì),近幾年一直位于STD的前三位。因此,如何有效控制和預(yù)防梅毒,已成為全世界普遍關(guān)注的公共衛(wèi)生問(wèn)題,而制備出具有完全保護(hù)作用的梅毒疫苗則是其中的關(guān)鍵。 目前,國(guó)內(nèi)外對(duì)Tp疫苗成分的研究主要集中于Tp重組外膜蛋白。Tp外膜蛋白既在Tp毒力中具有重要作用,也是宿主保護(hù)性免疫的主要靶標(biāo)。由于Gpd和Tp92外膜蛋白序列高度保守且與其它致病性密螺旋體有高度的同源性,以及其較強(qiáng)的免疫原性和保護(hù)性,均被認(rèn)為是研究Tp疫苗的最佳候選蛋白之一。本研究是在我們首次完成Tp單基因DNA疫苗篩選構(gòu)建及免疫活性初步研究工作的基礎(chǔ)上,將具有顯著免疫佐劑效應(yīng)的IL-2基因插入已真核表達(dá)重組體pcDNA3.1(+)/Gpd中,融合構(gòu)建多價(jià)DNA疫苗,用殼聚糖(CS)納米顆粒包被后導(dǎo)入新西蘭兔體內(nèi),利用CS納米顆粒高效的基因轉(zhuǎn)導(dǎo)能力及IL-2的佐劑效應(yīng)誘導(dǎo)動(dòng)物細(xì)胞表達(dá)免疫原性更強(qiáng)的融合蛋白,強(qiáng)化免疫應(yīng)答；同時(shí)采用核酸疫苗初免、含CpG序列的寡脫氧核苷酸(CpGODN)-Tp膜蛋白黏膜免疫加強(qiáng)的疫苗接種策略,以期全面激發(fā)機(jī)體免疫系統(tǒng),尤其黏膜免疫,建立高效抗Tp感染的動(dòng)物模型,為研制人用的高效疫苗預(yù)防梅毒打下基礎(chǔ)。 目的： 1.在篩選Tp外膜蛋白基因Gpd和Tp92的單基因核酸疫苗在新西蘭兔體內(nèi)免疫活性的基礎(chǔ)上,進(jìn)一步構(gòu)建Tp Gpd-IL-2基因融合殼聚糖納米核酸疫苗,證實(shí)該類疫苗能否有效轉(zhuǎn)染動(dòng)物細(xì)胞,表達(dá)融合蛋白,誘導(dǎo)機(jī)體產(chǎn)生特異性免疫應(yīng)答；證實(shí)細(xì)胞因子佐劑hIL-2和CS納米顆粒能否有效輔佐疫苗強(qiáng)化免疫應(yīng)答。 2.構(gòu)建CpG ODN-Tp Gpd-IL-2重組膜蛋白黏膜疫苗；在上述基礎(chǔ)上,應(yīng)用核酸疫苗肌注初免-CpG ODN-蛋白黏膜疫苗加強(qiáng)免疫的接種策略,驗(yàn)證該策略能否全面激發(fā)機(jī)體免疫應(yīng)答,特別是黏膜免疫應(yīng)答。 3.建立Tp感染的新西蘭兔動(dòng)物模型,確切評(píng)價(jià)核酸疫苗肌注初免-CpG ODN-蛋白黏膜疫苗加強(qiáng)免疫的接種策略的免疫保護(hù)效果,為最終建立能完全有效抗Tp感染的動(dòng)物模型,研制人用的高效疫苗打下良好基礎(chǔ)。 方法： 1. Tp Nichols的傳代與基因組提�。簩⒌蜏貎龃嫱貌G丸室溫解凍后剪碎置于無(wú)菌生理鹽水,讓睪丸內(nèi)保存的Tp Nichols株復(fù)蘇、釋放,再接種活兔睪丸傳代,恢復(fù)毒力及活性后用于Tp感染實(shí)驗(yàn)及TpDNA基因組提取。 2.真核與原核表達(dá)重組體的構(gòu)建：PCR釣取目的基因TpGpd、Tp92和hIL-2,與真核載體融合構(gòu)建pcDNA3.1(+)/Gpd、 pcDNA3.1(+)/IL-2及pcDNA3.1(+)/Gpd-IL-2多價(jià)真核表達(dá)重組體；將目的基因IL-2與已成功構(gòu)建的原核表達(dá)重組體(pET28a/Gpd)融合構(gòu)建多價(jià)原核表達(dá)重組體(pET28a/Gpd-IL-2);測(cè)序鑒定。 3.真核與原核表達(dá)重組體的表達(dá)與鑒定：免疫印跡法鑒定pcD/TpGpd、pcD/Tp92、pcD/IL-2及pcD/Gpd-IL-2真核表達(dá)重組體在體外哺乳動(dòng)物細(xì)胞內(nèi)的表達(dá)；pET28a/TpGpd-IL-2原核表達(dá)重組體的蛋白表達(dá)、純化、鑒定；將純化的重組蛋白抗原免疫動(dòng)物獲取多抗。 4. pcD/TpGpd和pcD/Tp92單基因核酸疫苗免疫活性與保護(hù)性研究：將實(shí)驗(yàn)用兔隨機(jī)分為pcD/Tp92疫苗組、pcD/TpGpd疫苗組、pcD空質(zhì)粒對(duì)照組和PBS對(duì)照組4個(gè)小組；肌肉多點(diǎn)注射免疫,每2w免疫1次,共免疫3次。并于初次免疫后第8w每組隨機(jī)取3只兔無(wú)菌分離脾細(xì)胞培養(yǎng),MTT法檢測(cè)兔脾淋巴細(xì)胞增殖水平,ELISA試劑盒檢測(cè)脾細(xì)胞培養(yǎng)上清IL-2及IFN-γ誘導(dǎo)水平,在免疫及感染期間不同時(shí)間點(diǎn)分別采用ELISA檢測(cè)免疫兔特異性抗體產(chǎn)生水平；第10w各實(shí)驗(yàn)組兔皮下接種TpNichols標(biāo)準(zhǔn)株進(jìn)行皮膚感染實(shí)驗(yàn),在感染后0-60d期間每隔3d觀察記錄感染部位皮損Tp陽(yáng)性率及潰瘍病灶發(fā)生率。 5.多基因疫苗的制備：制備CS納米顆粒包裹核酸疫苗；制備CpG ODN-Gpd-IL-2蛋白黏膜疫苗。 6.殼聚糖納米包裹核酸疫苗在新西蘭兔中免疫活性及保護(hù)性研究：實(shí)驗(yàn)用兔隨機(jī)分為12組,6個(gè)疫苗組(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個(gè)對(duì)照組(pcD/IL-2+CS, pcD/IL-2, pcD+CS, pcD, CS和PBS組),肌肉多點(diǎn)注射免疫,每隔2w加強(qiáng)免疫一次共3次,并于初次免疫后第8w每組隨機(jī)取3只兔無(wú)菌分離脾細(xì)胞培養(yǎng),MTT法檢測(cè)兔脾淋巴細(xì)胞增殖水平,ELISA試劑盒檢測(cè)脾細(xì)胞培養(yǎng)上清IL-2及IFN-γ誘導(dǎo)水平,在免疫及感染期間不同時(shí)間點(diǎn)分別采用ELISA檢測(cè)免疫兔特異性TpGpd抗體產(chǎn)生水平；第10w各實(shí)驗(yàn)組兔皮下接種TpNichols標(biāo)準(zhǔn)株進(jìn)行皮膚感染實(shí)驗(yàn),在感染后0-60d期間每隔3d觀察記錄感染部位皮損Tp陽(yáng)性率及潰瘍病灶發(fā)生率。比較、綜合分析各類疫苗誘導(dǎo)的免疫應(yīng)答水平及免疫保護(hù)效果。 7.核酸疫苗初免—蛋白黏膜疫苗加強(qiáng)免疫的接種策略評(píng)價(jià)：采用核酸疫苗肌注初免-CpGODN-蛋白黏膜疫苗加強(qiáng)免疫的疫苗接種策略,檢測(cè)新西蘭兔體內(nèi)細(xì)胞免疫和體液免疫應(yīng)答水平,評(píng)價(jià)免疫活性。對(duì)比評(píng)估核酸疫苗肌注初免—蛋白黏膜免疫加強(qiáng)的疫苗接種策略的抗Tp感染免疫保護(hù)效果。 結(jié)果： 1.成功復(fù)蘇、傳代Tp Nichols標(biāo)準(zhǔn)株并維持其良好的感染活性,可用于疫苗免疫后感染實(shí)驗(yàn)；成功制備Tp基因組DNA用于下一步研究。 2.構(gòu)建的真核重組質(zhì)粒pcD/Gpd-IL-2、pcD/Gpd、pcD/IL-2和pcD/Tp92經(jīng)酶切和測(cè)序鑒定證實(shí)插入片段為目的基因,測(cè)序結(jié)果與Genbank上登錄序列完全一致,均能在HeLa細(xì)胞內(nèi)有效表達(dá)靶蛋白；構(gòu)建的原核重組質(zhì)粒pET28a/Gpd-IL-2能在Rosetta表達(dá)菌有效表達(dá)一個(gè)相對(duì)分子量(Mr)約60KD的融合蛋白。 3.pcD/TpGpd和pcD/Tp92單基因疫苗均能在免疫兔體內(nèi)誘生比對(duì)照組(pcDNA3.1(+),PBS對(duì)照組)更高的抗TpGpd或Tp92特異性IgG抗體水平(P0.001),刺激更高的IL-2、IFN-γ細(xì)胞因子分泌(P0.001)及更強(qiáng)的脾細(xì)胞增殖分化(P0.001)。 4.pcD/Gpd-IL-2疫苗組和pcD/Gpd+pcD/IL-2聯(lián)合疫苗組均能在兔體內(nèi)誘生比pcD/Gpd單基因疫苗組更高的TpGpd特異性抗體水平(P0.05),刺激更高的IL-2、IFN-γ細(xì)胞因子分泌(P0.05)及更強(qiáng)的脾細(xì)胞增殖分化(P0.05)。 5.CS納米顆粒對(duì)pcD/Gpd+pcD/IL-2聯(lián)合疫苗組、pcD/Gpd-IL-2疫苗組及pcD/Gpd單基因疫苗的包裹未能顯著促進(jìn)相應(yīng)疫苗組在兔體內(nèi)誘生更高的的特異性抗體水平(P0.05),未能刺激更高的IL-2、IFN-γ細(xì)胞因子分泌(P0.05)及更強(qiáng)的脾細(xì)胞增殖分化(P0.05)。 6.Tp的兔皮膚感染導(dǎo)致感染期內(nèi)各對(duì)照組特異性抗體水平及脾細(xì)胞增殖分化較之感染前有著顯著升高(P0.05),對(duì)各疫苗組有一定促進(jìn)作用；IL-2基因佐劑對(duì)各Tp Gpd DNA疫苗在感染期兔體內(nèi)特異性抗體水平及脾細(xì)胞增殖分化的長(zhǎng)久促進(jìn)及維持有顯著作用,而CS納米顆粒對(duì)各Tp Gpd DNA疫苗的包裹未能顯著提高在感染期階段兔體內(nèi)特異性抗體水平及脾細(xì)胞增殖分化。 7.各Tp Gpd DNA疫苗相比各對(duì)照組均能顯著降低Tp感染部位皮損Tp陽(yáng)性率及潰瘍病灶形成率(P0.001),顯示出較強(qiáng)的免疫保護(hù)效應(yīng)；pcD/IL-2基因佐劑較之CS納米顆粒更能增強(qiáng)pCD/TpGpd疫苗的免疫保護(hù)效果；兩者聯(lián)合使用可達(dá)到較之其它疫苗組更好的免疫效果。 8.在各實(shí)驗(yàn)組兔背部皮膚8個(gè)位點(diǎn)皮內(nèi)Tp感染后0-60d同比觀察比較,用CS+pCD/Gpd+pcD/IL-2聯(lián)合疫苗和pCD/Gpd+pcD/IL-2聯(lián)合疫苗,CS+pcD/Gpd-IL-2疫苗和pcD/Gpd-IL-2疫苗免疫兔后感染位點(diǎn)皮損紅腫相對(duì)其它實(shí)驗(yàn)組直徑最小,潰瘍形成率最少,也最早愈合(42-45d),各組間差異不顯著；PcD/Gpd和CS+pcD/Gpd疫苗組兔后感染位點(diǎn)皮損皮損紅腫直徑同比中等大小,兩組間差異不顯著；各對(duì)照組(CS+pcD/IL-2,pcD/IL-2,CS+pcD, pcD, CS+PBS,PBS對(duì)照組)感染位點(diǎn)皮損紅腫直徑最大,潰瘍病灶形成數(shù)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蛋白鼻飼加強(qiáng)免疫的接種策略既能刺激較強(qiáng)的體液和細(xì)胞免疫效應(yīng),與pcD/Gpd-IL-2疫苗肌注免疫組無(wú)顯著差異,還能刺激較高的黏膜免疫效應(yīng),導(dǎo)致最低Tp感染部位皮損Tp陽(yáng)性率(0%)及潰瘍病灶形成率(3.33%)從而達(dá)到更有效地保護(hù)作用。 結(jié)論： 1.Tp92和TpGpd單基因DNA疫苗組經(jīng)肌肉注射免疫新西蘭兔均可誘生較強(qiáng)的體液免疫和細(xì)胞免疫應(yīng)答,產(chǎn)生較好的保護(hù)作用。 2.IL-2基因與Tp Gpd抗原基因無(wú)論是融合表達(dá)還是非融合表達(dá)均能顯著增強(qiáng)TpGpd單基因疫苗的免疫效應(yīng)和免疫保護(hù)作用。 3.CS納米顆粒包裹Tp Gpd DNA疫苗對(duì)疫苗的體液及細(xì)胞免疫效應(yīng)及抗Tp皮膚感染有一定促進(jìn)作用,但效果不顯著。而CS納米顆粒包裹加上IL-2基因佐劑的聯(lián)合應(yīng)用則能刺激pcD/Gpd疫苗產(chǎn)生更強(qiáng)的免疫效應(yīng)及更好的抗Tp皮膚感染效果。 4.采用pcD/Gpd-IL-2疫苗肌注初免,CpGODN+Gpd-IL-2蛋白鼻飼加強(qiáng)免疫的接種策略,既能刺激機(jī)體產(chǎn)生較強(qiáng)的體液和細(xì)胞免疫效應(yīng),還能刺激較強(qiáng)的黏膜免疫效應(yīng),激發(fā)更有效的免疫保護(hù)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 趙飛駿,吳移謀,劉雙全,余敏君;Construction of the Eukaryotic Expression Vector for Outer Membrane Protein Tp92 from Treponema pallidum and Its Preliminary Study on the Immune Responses in New Zealand Rabbits[J];Journal of Microbiology and Immunology;2004年03期

2 ;Eukaryotic expression of outer membrane protein Gpd from Treponema pallidum and preliminary studies on its immune response in rabbits[J];Journal of Microbiology and Immunology;2005年02期

3 劉雙全;吳移謀;;梅毒螺旋體4種主要膜蛋白分子的研究現(xiàn)狀[J];國(guó)外醫(yī)學(xué)(微生物學(xué)分冊(cè));2004年01期

4 曾鐵兵;裴瑞青;張躍軍;嚴(yán)加林;劉雙全;趙飛駿;吳移謀;;梅毒螺旋體Tp0993重組蛋白的表達(dá)及鑒定[J];南華大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年05期

5 ;Production of proinflammatory cytokines in the human THP-1 monocyte cell line following induction by Tp0751,a recombinant protein of Treponema pallidum[J];Science China(Life Sciences);2010年02期

6 劉雙全;汪世平;吳移謀;趙飛駿;曾鐵兵;張躍軍;張秋桂;高冬梅;;梅毒螺旋體Tp0751重組蛋白誘導(dǎo)人單核細(xì)胞表達(dá)前炎癥細(xì)胞因子的研究[J];中國(guó)科學(xué):生命科學(xué);2010年01期

7 ;LT(K63/R72),a New Mutant of Escherichia coli Heat-labile Enterotoxin,Exhibits Characteristics More Similar to LT(K63)than LT(R72)[J];Acta Biochimica et Biophysica Sinica;2005年02期

8 陳創(chuàng)夫,余興龍,馬正海,李紅衛(wèi),李作生,涂長(zhǎng)春,殷震;豬瘟基因疫苗在小鼠肌肉中的免疫病理學(xué)觀察[J];石河子大學(xué)學(xué)報(bào)(自然科學(xué)版);2001年03期

9 ;Regulating Effects of Novel CpG Chitosan-nanoparticles on Immune Responses of Mice to Porcine Paratyphoid Vaccines[J];Biomedical and Environmental Sciences;2006年04期

10 張以芳;核酸疫苗的構(gòu)成及主要特點(diǎn)[J];上海畜牧獸醫(yī)通訊;2000年02期

相關(guān)博士學(xué)位論文 前1條

1 張良芬;編碼梅毒螺旋體47-、17-和15kD脂蛋白的基因克隆、表達(dá)及表達(dá)產(chǎn)物反應(yīng)原性的初步鑒定[D];中國(guó)協(xié)和醫(yī)科大學(xué);2001年

，

本文編號(hào)：2205453