牛分枝杆菌三价组合及融合DNA疫苗免疫效果的研究

分泌性蛋白Ag85B、MPB64和ESAT-6为Mycobacterium boris的主要保护性抗原,在诱导机体免疫反应和抵抗感染中发挥重要作用。本研究以peDNA3．1（＋）为载体,构建了由上述3种抗原基因构成的不同疫苗：3基因融合（peDNA-MPB64-Ag85B-ESAT-6,pCMAE）DNA疫苗和三价（pcDNA-Ag85B＋peDNA-MPB64＋pcDNA-ESAT-6）DNA疫苗,评价各种DNA疫苗诱导的体液免疫、细胞免疫及以BCG攻毒后的免疫保护水平。试验结果表明,多基因融合DNA疫苗免疫后的小鼠血清抗体水平、淋巴细胞增殖（SI值）、gamma interferon（IFN-γ）和interleukin-2（IL-2）水平明显高于其他各组（P〈0．05）,攻毒保护效果也优于多价DNA疫苗,达到了BCG疫苗的免疫保护水平,表明本研究制备的融合DNA疫苗具有良好的应用前景。     

牛分枝杆菌二价组合及融合DNA疫苗的免疫效果

利用PCR技术和重叠延伸剪接(SOE)技术扩增牛分枝杆菌ag85b、mpb64基因和ag85b-mpb64融合基因,连接真核表达载体pCDNA3.1(+),构建二基因融合(pCDNA-MPB64/Ag85B,pCMA)和二价组合(pCDNA-Ag85B+pCDNA-MPB64,pCA+pCM)DNA疫苗.以牛分枝杆菌卡介苗(BCG)为阳性对照,以pCDNA3.1(+)和PBS为阴性对照,免疫BALB/c小鼠,检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN-γ及IL-2分泌情况.结果显示:融合DNA疫苗组免疫小鼠血清抗体水平、刺激值(SI值)及IFN-γ和IL-2的分泌水平均明显高于其他各组(P<0.05).二价组合DNA疫苗组的体液和细胞免疫指标与BCG组相当(P>0.05),而显著高于两阴性对照组(P<0.05).     

结核分支杆菌MTB41 DNA疫苗的构建及原核表达

为了构建结核分支杆菌MTB41 DNA疫苗,同时将其克隆到原核表达载体中进行表达。用结核分支杆菌H37Rv株基因组DNA为模板,用PCR法对基因MTB41进行扩增,克隆到真核表达载体pJW4303中,构建MTB41重组真核质粒(DNA疫苗),进行了酶切鉴定和序列分析;克隆到原核表达载体pET22b中,酶切和测序鉴定正确后转入E．coli BL21(DE3)PLysS宿主菌株内中,诱导表达,进行SDS-PAGE电泳分析。电泳发现转化了重组质粒的菌株有蛋白表达,所表达的蛋白质相对分子质量为41ku,MTB41 DNA疫苗酶切鉴定和序列分析完全正确,确定含有正确的读码框,疫苗制备成功。DNA疫苗的成功构建,重组蛋白MTB41的成功表达,为结核病诊断、重组疫苗应用和免疫效应检测以及抗原、抗体的大规模制备打下了基础。     

牛分枝杆菌esat-6和mpb70-mpb83基因DNA疫苗的免疫效果

用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因，连接真核表达载体pcDNA3．1（＋），构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠：pCE6组、pC70—83-E6组、pcDNA3．1（＋）和PBS对照组，采用间接EI。ISA法检测免疫小鼠血清特异性抗体水平，MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN—y分泌情况。结果表明，2重组质粒免疫组小鼠的血清抗体水平持续上升，而2对照组始终维持在较低水平，且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后，pCE6组和pC70—83-E6组小鼠的SI值与2对照组均差异显著（P〈0．05），2重组质粒免疫组间差异不显著（P〉0．05）；2重组质粒免疫小鼠脾细胞产生的IFN—y均显著高于2对照组（P〈0．05），且pC70-83-E6组明显高于其他3组（P〈0．05）。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。     

结核分枝杆菌分泌蛋白ESAT-6、CFP10基因的克隆、鉴定及其原核表达

以人型结核分枝杆菌 H37RV株基因组 DNA为模板 ,应用 PCR法对 ESAT- 6和 CFP10基因进行扩增 ,产物经纯化后与载体 PMD18- T连接、转化及酶切鉴定 ,亚克隆到原核表达载体 PGEX- 6 P- 1,构建原核重组表达质粒 ,转化入大肠杆菌 BL2 1中 ,以 1mmol/L IPTG诱导 ,进行 SDS- PAGE电泳。结果表明 ,ESAT- 6和 CFP10基因表达的融合蛋白相对分子质量分别为 32 0 0 0和 36 0 0 0 ,与实测相符。重组结核杆菌分泌蛋白 ESAT- 6和 CFP10的成功表达为结核病诊断及重组疫苗的构建打下了基础     

DNA疫苗的研究进展

DNA疫苗主要是由保护性抗原基因和质粒载体两部分组成。(一)编码抗原蛋白的基因可以是单个基因，也可以是具有协同保护功能的一组基因，也可以是编码抗原决定簇的一段核苷酸序列。如果对病原体基因组了解得较为清楚，就可以选择病原体表面糖蛋白编码基因，被表达的蛋白质可以在寄主体内正确地糖基化，从而诱导免疫反应。对于易发生变异的病毒，则可以选择各亚型共有的核心蛋白基因中的保守DNA序列作为编码抗原的序列，从而避免易变病毒产生免疫逃避现象。     

减毒沙门氏菌为载体传递DNA疫苗诱导对新城疫病毒的免疫保护

探讨了以减毒鼠伤寒沙门氏茵为栽体传递新城疫病毒DNA疫苗的安全性、免疫原性和可行性。将含新城疫病毒(NDV)F48E9株融合蛋白(F)基因的真核表达质粒pcDNA3-F的重组减毒鼠伤寒沙门氏菌ZJ111株(ZJ111／pcD-NA3一F菌株)，以10^8CFU进行首免，2周后二免，三免后4周攻击强毒株F48E9，观察其安全性和免疫原性，同时设只含空载体pcDNA3的ZJ111／pcDNA3菌株对照及口服PBS对照。结果表明：重组ZJ111／pcDNA3-F菌株具有良好的安全性。对强毒株攻击的保护率达64．7％。重组ZJ111／pcDNA3-F菌株不仅能诱导雏鸡产生NDVELISA抗体，而且诱导产生的法氏囊B淋巴细胞和胸腺T淋巴细胞增殖反应显著高于ZJ111／pcDNA3时照组。这些结果提示，减毒沙门氏菌为载体不仅可直接将NDVF基因呈递给鸡体细胞进行表达，产生抗NDV的体液免疫，而且还可诱导细胞免疫应答。     

结核分枝杆菌CFP10-ESAT-6融合基因的表达及其多克隆抗体的制备

以结核分枝杆菌标准株H37Rv的基因组DNA为模板，经重叠PCR扩增获得CFP10-ESAT-6融合基因片段，克隆于pET21a表达载体中，获得了重组质粒pET21a—CFP10—ESAT—6，将其转化大肠杆菌BL21细胞，经IPTG诱导表达，得到了25ku的目的蛋白。表达产物经纯化和定量分析后免疫新西兰大白兔，收集兔血清进行抗体ELISA检测。结果显示，成功构建了CFP10-ESAT-6融合基因的原核表达质粒，获得了CFP10-ESAT-6融合蛋白，以此蛋白免疫动物可产生高效价的抗体。     

结核分支杆菌HSP65 DNA疫苗的制备和重组蛋白的原核表达

结核分支杆菌分子量为65ku的热应激蛋白(HSP65)是一种非常重要的抗原，为了研制结核病核酸疫苗，构建编码HSP65DNA，并将其分别克隆到原核和真核载体中进行了表达。以标准结核分支杆菌H37Rv基因组DNA为模板，用PCR法扩增出HSP65基因，经限制性内切酶消化后，插入真核表达栽体pJW4303中，获得重组质粒pJW-HSP65。同时将HSP65基因插入原核表达栽体pET-22b( )，获得重组质粒pET22b-HSP65。将pET22b-HSP65重组质粒转化大肠杆菌蛋白酶缺陷型菌株BL21(DE3)／PolysS，用IPTG诱导，进行蛋白表达。结果表明，经酶切鉴定和序列测定证实插入片断为目的基因HSP65，构建成功了真核重组质粒pJW-HSP65即可作为结核病DNA疫苗。经SDS-PAGE检验证明可以在大肠杆菌细胞中高效表达，将表达蛋白进行纯化，作为保护性结核杆菌抗原以便检测HSP65DNA疫苗的免疫效果。     

DNA疫苗的特点及在不同动物中的应用

1特点DNA疫苗又称核酸疫苗或基因疫苗,是编码免疫原或与免疫原相关的真核表达质粒DNA（或RNA）。可经一定途径进入动物体内,被宿主细胞摄取后转录和翻译,表达出抗原蛋白,此抗原蛋白能刺激机体产生非特异性和特异性免疫应答反应,从而起到免疫保护作用。     

Immunogenic and protective effects of a DNA vaccine for Mycobacterium marinum in fish

Mycobacteriosis, caused by numerous Mycobacterium spp., can be a devastating disease of both wild and cultured fishes. As no efficacious treatment exists, a vaccine against fish mycobacteriosis is essential for prevention and control of this disease. Thus, a DNA vaccine was constructed using the Mycobacterium marinum Ag85A gene that encodes one of the major secreted fibronectin-binding proteins of Mycobacterium spp., which was isolated and then subcloned into a commercially available eukaryotic expression vector. Juvenile hybrid striped bass (Morone saxatilis x M. chrysops), a species known to be particularly susceptible to this disease, were immunized by i.m. and i.p. injection with the resulting construct and as a result produced specific immune responses towards the Ag85A. Increasing concentrations of humoral antibodies to the Ag85A antigen were generated in all DNA vaccine groups, while macrophage phagocytosis and respiratory burst functions failed to exhibit upregulation after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to a live M. marinum challenge 90 days post-inoculation, as demonstrated by increased survival of vaccinated fish over control fish and by reduced splenic bacterial counts in vaccinated fish. Furthermore, humoral immune responses and protective effects were significantly increased at higher vaccine doses using the i.m. injection route.     

牛分枝杆菌CFP-10和ESAT-6基因的融合表达及其抗原活性鉴定

CFP-10和ESAT-6是牛分枝杆菌的免疫优势抗原,可诱导机体产生IFN-γ,在牛结核病的免疫诊断中发挥重要作用。本试验分别克隆了牛分枝杆菌CFP-10和ESAT-6基因,并将这2个基因融合扩增,构建重组表达质粒pET28a/CFP-10-ESAT-6,重组质粒在大肠杆菌BL21中进行IPTG诱导表达。重组蛋白主要以分泌表达的形式存在于表达上清中,收集上清中的目的蛋白进行Ni亲和层析柱和分子筛两步纯化,蛋白纯度分别达到90%和95%。将纯化的重组蛋白以2mg/L的质量浓度包被酶标反应板,用间接ELISA方法检测不同来源牛血清中的结核病抗体,结果表明,表达的重组融合蛋白具有良好的抗原活性,可有效识别阴、阳性结核病血清。     

Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens

A novel Salmonella Enteritidis (SE) vaccine candidate strain, JOL919 was constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. The study was carried out to evaluate the strain as a vaccine candidate against salmonellosis. The strain showed the enhanced macrophage invasion, early bacterial clearance and higher immune responses as compared to the other mutants, JOL917 (Δlon) and JOL918 (ΔcpxR), and the wild type. In further analysis, the chickens immunized with JOL919 showed a significant increase in plasma IgG and intestinal secretory IgA levels, which was an indication of robust humoral and mucosal immune responses induced by the candidate. The lymphocyte proliferation response and CD45(+)CD3(+) T cells, associated with an activation of T helper and cytotoxic cells, were also significantly increased in the immunized group, which indicated that the candidate also induced cellular immune responses. The immune cell influx into caecal tissues analyzed by immunohistochemistry showed that CD8(+) T cells were predominated in the immunized group, suggesting that the candidate can clear the invaded pathogen in the intestines by a more direct way involving cytotoxic activity. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.     

Field evaluation of the protective efficacy of Mycobacterium bovis BCG vaccine against bovine tuberculosis

The protective efficacy of Mycobacterium bovis BCG (1 × 10⁶ single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNγ PPD-B and IFNγ ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age.     

牛分枝杆菌抗原MPB70、MPB83和ESAT-的融合表达及重组蛋白的初步应用

应用PCR方法从牛分枝杆菌Vallee株基因组中扩增获得mpb70、mpb83和esat-6三个目的基因片段。采用重叠延伸剪接技术（splicing by overlap extension，SOE）获得融合基因mpb70-mpb83后，将mpb70-mpb83和esat-6串连于同一表达载体pET32a（＋）中得到重组质粒pET70-83-E6。转化BL21（DE3）大肠杆菌感受态后，经IPTG诱导以可溶的形式表达融合蛋白。用Ni^2＋亲合层析的方法纯化该融合蛋白。Western blot分析显示：该融合蛋白能与抗牛分枝杆菌阳性血清发生特异性反应，而与牛副结核病阳性血清不反应。用该纯化蛋白初步建立了间接ELISA方法，并检测了117份临床血清样本（其中67份为PPD阳性牛血清），阳性率为39．32％（46／117）份，与PPD皮试诊断的符合率为82．05％（96／117）。     

牛分枝杆菌抗原MPB70、MPB83和ESAT-6的融合表达及重组蛋白的初步应用

应用PCR方法从牛分枝杆菌Vallee株基因组中扩增获得mpb70、mpb83和esat-6三个目的基因片段。采用重叠延伸剪接技术(splicing by overlap extension,SOE)获得融合基因mpb70-mpb83后,将mpb70-mpb83和esat-6串连于同一表达载体pET32a( )中得到重组质粒pET70-83-E6。转化BL21(DE3)大肠杆菌感受态后,经IPTG诱导以可溶的形式表达融合蛋白。用Ni2 亲合层析的方法纯化该融合蛋白。Western blot分析显示:该融合蛋白能与抗牛分枝杆菌阳性血清发生特异性反应,而与牛副结核病阳性血清不反应。用该纯化蛋白初步建立了间接ELISA方法,并检测了117份临床血清样本(其中67份为PPD阳性牛血清),阳性率为39.32%(46/117)份,与PPD皮试诊断的符合率为82.05%(96/117)。     

Improving protective efficacy of BCG vaccination for wildlife against bovine tuberculosis

Possums are a wildlife vector of bovine tuberculosis in New Zealand. Vaccination of possums with BCG is being considered as a measure to control the spread of bovine tuberculosis to cattle and deer. Delivery via oral bait is feasible but BCG is degraded in the stomach. The aim was to determine whether ranitidine (Zantac) would reduce gastric acidity and enhance the efficacy of intragastrically administered BCG. A dose of 75 mg reduced gastric acidity for at least 4 h. Thus, possums were vaccinated intragastrically with BCG after receiving 75 mg ranitidine or ranitidine or BCG alone, as controls, before challenge with virulent Mycobacterium bovis. Proliferative responses of blood lymphocytes to M. bovis antigens after vaccination were significantly higher in possums given ranitidine/BCG compared to controls and seven weeks after challenge they had significantly lower lung weights and spleen bacterial counts than ranitidine alone controls. Vaccination with BCG alone only gave a reduction in loss in body weight. Agents that reduce gastric acidity may be useful in formulating BCG for oral bait delivery to wildlife for vaccination against bovine tuberculosis.     

猪圆环病毒2型灭活疫苗最小免疫保护剂量研究

猪圆环病毒2型(PCV2)是引起断奶仔猪多系统衰竭综合征(PMWS)的主要病原,疫苗是防制该病的主要手段.为研究PCV2灭活疫苗的免疫效果,本研究将PCV2 SH株灭活,用生理盐水稀释成5×105.0TCID50/mL(高剂量)、2.5×1050 TCID50/mL(中等剂量)、1.25×105.0 TCID50/mL(低剂量)、6.3×104.0 TCID50/mL(超低剂量)4种剂量,与等量白油佐剂乳化后,通过断乳仔猪的免疫攻毒试验观察该灭活疫苗的免疫特性.结果表明,PCV2疫苗抗原含量大于1.25×105.0 TCID50/mL时免疫仔猪后均可以产生特异性抗体,能够明显减轻攻毒后仔猪的临床症状、缓解组织病变、降低病毒血症,对免疫猪有一定的保护效果,可以作为预防PCV2感染的疫苗使用.本研究为研制商品化的PCV2灭活疫苗提供实验依据.