MM-7基因工程双价疫苗对新疆细毛羊免疫效果的研究

应用ＭＭ－７基因工程双价疫苗免疫产前１５天的新疆细毛孕母羊，并通过改变产前１５天孕母羊机体免疫水平，由初乳被动免疫羔羊。在免疫孕母羊后１０，２０，３０，４０，５０，６０，７０天分别检测免疫组及对照组血清特异性中和抗体ＩｇＧ及ＩｇＡ动态水平，观察两组腹泻发病率，死亡率情况和ＭＭ－Ｕ基因工程双价疫苗的免疫保护效果。     

微小隐孢子虫CP23 DNA疫苗免疫保护作用研究

目的研究微小隐孢子虫表面抗原CP23 DNA疫苗产生的免疫反应及对小鼠的免疫保护作用。方法选BALB/c小鼠60只,随机分为3组,即疫苗免疫组、PBS对照组及空质粒对照组。疫苗免疫组用构建的真核表达质粒pcDNA 3.0-23每隔2周免疫1次小鼠,共免疫3次。PBS对照组肌注等量PBS,空质粒对照组肌注pcDNA3.0,免疫方法及次数同疫苗免疫组。末次免疫2周后,分别取小鼠脾脏、血清,检测CD4+和CD8+T细胞、细胞因子IFN-γ及抗CP23特异性抗体IgG和IgA滴度;用微小隐孢子虫攻击感染被免疫小鼠,收集小鼠粪便,计算小鼠排出的卵囊量。结果 PBS对照组及空质粒组比较,疫苗免疫组小鼠的CD4+T细胞、CD4+/CD8+比值及脾细胞培养上清中IFN-γ滴度均显著升高(P0.05),小鼠血清抗CP23特异性抗体IgG及IgA滴度随免疫次数增加显著升高(P0.05),攻击感染后小鼠排卵囊量显著减少(P0.05),且排出时间缩短。结论构建的微小隐孢子虫表面抗原CP23真核表达质粒能够诱导小鼠产生细胞及体液免疫反应,对小鼠有较好的免疫保护作用。     

轮状病毒NSP4基因在非复制型腺病毒载体中的表达及免疫活性

目的:研究以非复制型重组腺病毒表达人轮状病毒NSP4基因作为新型轮状病毒基因疫苗的可行性．方法:用AdEasy系统构建表达NSP4蛋白的重组腺病毒,并通过形态学观察、PCR、RT- PCR、Southern blot和Western blot等对重组腺病毒进行鉴定．通过滴鼻途径免疫♀小鼠,对免疫后母鼠生产的乳鼠(4日龄)用SA11株轮状病毒灌胃攻击,观察腹泻的产生情况并进行腹泻评级．结果:获得了重组腺病毒rvAdEasyNSP4． Southern blot表明,在重组腺病毒中确有特异性的NSP4基因整合,Western blot证明,目的蛋白得到表达．初次免疫小鼠后血清IgG抗体的滴度可达1:1 000,但阳转率仅为28．5％;再次免疫后,血清抗体阳转率达到了85．7％;第3次免疫后,血清抗体阳转率达到100%．血清IgA 阳转率可以达到71．4％．实验组乳鼠攻毒后腹泻的百分率和腹泻评分(反应腹泻严重程度) 均较对照组低．结论:腺病毒载体可有效表达轮状病毒非结构蛋白NSP4并可有效诱导小鼠免疫反应,这些结果的取得为新型轮状病毒疫苗的研制提供了依据．     

人抗SARS-CoV-2感染适应性免疫应答的研究进展

由SARS-CoV-2感染引起的COVID-19为全球流行病,严重威胁全球公共卫生,感染无特异性免疫力人群影响人们改变社交行为和习惯,产生全球经济负担,目前尚无有效治疗药物。因此,自然感染诱导的适应性免疫应答备受关注。自然感染SARS-CoV-2可诱导绝大多数成人产生体液免疫应答和细胞免疫应答,形成高滴度S蛋白特异性IgM、IgG和IgA类抗体、高水平中和抗体、强CD4⁺T细胞应答、特异性记忆B细胞和记忆T细胞,可获得抗SARS-CoV-2感染的免疫保护,但免疫保护持续时间仍不确定。对SARS-CoV-2感染诊断、血清学监测、免疫策略、感染防控及疫苗开发等有重要意义。     

日本血吸虫97kDa DNA疫苗与致弱尾蚴疫苗诱导免疫应答特征的比较研究

目的　对比观察日本血吸虫大陆株副肌球蛋白基因疫苗(Sjc97DNA)与紫外线致弱尾蚴(UVC)疫苗免疫C57BL6小鼠诱导的抗感染保护力及免疫应答特征。　方法　以Sjc97DNA核酸疫苗经后腿胫前肌免疫C57BL6小鼠共2次,每次间隔3wk,末次免疫后3wk攻击感染日本血吸虫尾蚴;UVC疫苗接种同种小鼠后5wk攻击感染上述等量尾蚴。均于攻击感染后7wk计数虫负荷及肝卵负荷。并设空质粒对照及感染对照组。用ELISA分析免疫鼠攻击感染前后血清特异性IgG、IgA及亚型抗体水平,以及脾淋巴细胞体外诱生的细胞因子水平。　结果　Sjc97DNA疫苗及UVC疫苗免疫小鼠均诱生出以Th1型免疫应答为主的IL2、IFNγ及特异性抗AWA、SEAIgG2a、IgG2b亚型及IgA抗体,UVC疫苗组小鼠各细胞因子及抗体水平均显著高于Sjc97DNA疫苗组,但两疫苗组均未测及IL4。攻击感染后,Sjc97DNA疫苗组的减虫率36.3%、减卵率42.4%,明显低于UVC疫苗组的66.9%和75.6%。攻击感染后7wk,两疫苗组小鼠Th2型免疫应答虽有所增强,但仍以Th1型免疫应答占优势;而空质粒对照组和感染对照组小鼠则以Th2型免疫应答为主。　结论　核酸疫苗与紫外线致弱尾蚴疫苗均能诱导产生抗感染免疫保护力,致弱尾蚴疫苗的免疫保护力高于Sjc97DNA。两疫苗诱导的抗感染     

A组牛轮状病毒基因工程亚单位疫苗的初步研究

目的构建轮状病毒外膜蛋白VP4、VP7基因与大肠杆菌LTB蛋白基因的重组表达载体,研究重组表达蛋白的免疫原性,为研制犊牛腹泻基因工程亚单位疫苗奠定基础。方法克隆A组牛轮状病毒临床分离株CHLY的VP4、VP7基因和大肠杆菌LTB基因,插入pET32a中构建了4个原核表达载体pET32a／VP7、pET32a／VP4、pET32a／VP7-LTB、pET32a／VP4-LTB,转化到BL21（DE3）中进行表达纯化,纯化蛋白与弗氏佐剂混合免疫10w龄雌鼠,在实验期间对雌鼠进行交配,对其所产乳鼠用150μL10^-6.5／0．1mL TCID20的轮状病毒进行攻毒以研究重组蛋白免疫原性和攻毒保护性。结果重组蛋白在大肠杆菌BL21（DE3）中以包涵体的形式存在,Bandscan扫描分析结果显示,它们分别占菌体总蛋白的35％、20％、30％、18％,利用Ni-NTA Agarose在变性条件下成功纯化得到高纯度重组蛋白。10w龄雌鼠免疫试验表明rVP4-LTB组激发了最强的免疫反应,rVP4组次之,rVP7-LTB组略高于rVP7,各个免疫组问的差异性不显著（P〉0．05）,各实验组IgG抗体水平显著高于空质粒对照组（P〈0．01）。雌鼠所产乳鼠的攻毒实验表明,免疫组雌鼠所产乳鼠腹泻率为13．35％～23．8％,腹泻持续时间为48h～72h,较空质粒对照组明显降低,腹泻症状较轻且症状持续时间明显缩短。四个重组蛋白组攻毒保护率为76．2％～86．7％,rVP7-LTB、rVP4-LTB两组略高于rVP7、rVP4组,但相互间差异不显著（P〉0．05）。结论重组蛋白具有较好的免疫原性,被免疫雌鼠体内产生的中和性抗体可以经被动免疫过程传递至子代,并为仔鼠提供保护。本研究为犊牛轮状病毒基因工程疫苗的研究奠定了一定的基础。     

日本血吸虫糖基诱导抗体类型与感染状态的关系研究

目的以日本血吸虫感染猪为模型,研究不同感染状态下针对糖基产生的抗体在宿主免疫应答中的作用。方法建立日本血吸虫感染猪模型,分别检测不同感染状态下猪血清中针对LNFPIII、N-糖苷水解酶F(PNGaseF)处理可溶性虫卵抗原(SEA)前后及过碘酸处理SEA前后的抗体水平。结果辐照致弱尾蚴单纯免疫组、正常尾蚴单纯感染组和免疫后予以感染的免疫攻击组的猪均针对LNFPIII产生了相应的IgM抗体应答。3组猪血清中针对PNGaseF处理SEA的特异性抗体IgG、IgM水平较未处理组略低,但抗体IgG和IgM水平差异均无统计学意义(P均0.05)。在单纯免疫组,猪血清中针对过碘酸处理SEA的特异性IgG、IgM、IgA抗体水平均较未处理组有所下降,差异有统计学意义(P均0.01),但辐照致弱日本血吸虫糖基成分所诱导的抗体水平均较低。在单纯感染组,猪血清中针对过碘酸处理SEA的抗体IgG、IgM水平在感染后第8周下降极为显著,抗体水平差异有统计学意义(P均0.01),其中糖基所诱导的抗体亚型以IgG1为主。此时,在虫负荷显著降低的免疫攻击组,日本血吸虫糖基成分能够显著增强IgG、IgM、IgA,特别是IgG2的表达。结论日本血吸虫糖基成分能够诱导宿主产生多种类型的抗体应答;且抗体亚型的表达与宿主的感染状态相关,即IgG1亚型的表达与日本血吸虫急性感染期的虫负荷和卵负荷一致;而具有免疫保护力的宿主能够针对日本血吸虫糖基成分产生IgG2应答。     

空肠弯曲菌28-31 ku外膜蛋白的免疫原性

目的:探讨空肠弯曲菌28-31 ku外膜蛋白的免疫原性. 方法:将BALB/c小鼠分为正常对照组和不同剂量抗原的免疫组,正常对照组不予抗原免疫,而免疫组采用28- 31 ku蛋白(纯)或甘氨酸提取的外膜蛋白(粗),分别加用或不加用完全弗氏佐剂(F),采用sc,在0,1,2,3, 4 wk免疫,于末次免疫后10 d,用双向免疫琼脂扩散试验测定各免疫组抗血清效价,待效价达到1:4至1:16 时.分别采集各免疫组及对照组小鼠的血清、空肠及回肠内的肠液,用间接ELISA法检测各组标本的特异性抗体IgA,IgG. 结果:不同剂量的抗原sc免疫BALB/c小鼠后,免疫组血清及肠液中特异性IgA,IgG抗体水平较正常对照组及实验对照组显著升高(P<0.05). 结论:空肠弯曲菌的28-31 ku外膜蛋白是一种良好的免疫原,能够诱导BALB/c小鼠产生特异性抗体,可能成为空肠弯曲菌亚单位疫苗候选的重要组分.     

HIV—1CN54合成gp120基因（syngp120）的DNA疫苗鼻内免疫小鼠诱导全身的和粘膜的免疫应答

目的 初步探讨HIV－1CN54合成gp120基因的DNA疫苗（pcDNA3．1－syngp120）鼻内接种小鼠否诱发免疫应答。方法 DNA疫苗免疫后，制备脾和肠系膜淋巴结（MLN）淋巴细胞，在体外测其增殖应答和CD8^ CTL应答。间接ELISA法测血清和粘膜洗液抗原－特异的IgG和IgA抗体滴度。中和实验免疫血清和阴道洗液是否和HIV－1SF33。结果 在DNA疫苗末次免疫后，小鼠第1周检测到脾和MLNCD8^ CTL应答较弱，而第5周末检测到。另外，在末次免疫后的第1、5周检测到MLN而未检测到脾淋巴细胞发生增殖应答。并且检测到特异的血清IgG抗体和粘膜的（包括粪便和阴道洗液）IgA抗体，但未检测到血清的IgA抗体和粘膜的（包括粪便和阴道洗液）IgG抗体。中和实验发现末次免疫后第5周的血清能中和实验室毒株HIV－1SF33（B亚型），而阴道洗液则没有。结论 该DNA疫苗鼻内免疫小鼠可诱导粘膜免疫应答，包括MLN淋巴细胞增殖应答和CD8^ CTL应答，同时诱导较弱的粘膜IgA抗体应答。此外，能诱导脾CD8^ CTL应答和血清IgG抗体应答。免疫血清中和HIV－1SF33，而阴道洗液不能。     

减毒沙门氏菌投递的TGEV S基因疫苗口服免疫猪的动态规律

目的 研究TGEV S基因疫苗SL7207(PVAXD-TS)口服免疫仔猪后的动态免疫诱导规律。方法 将口服疫苗SL7207(PVAXD-TS)、空载体菌株SL7207(PVAXD)以1.6×10¹¹CFU/头的剂量口服接种20日龄仔猪,以PBS为空白对照,2周后以2.0×10¹¹CFU的剂量加强免疫。分别测定0、2、4、6和8周的血清IgG、IgA、TGEV中和抗体、细胞免疫反应 IL-4、IFN-γ、外周血淋巴细胞增殖情况。结果 仔猪口服免疫后第4周即可检测抗TGEV的特异性IgG和粪黏液IgA抗体;在免疫后第6周IgA、IgG、IL-4、IFN-γ抗体和外周血T淋巴细胞增殖与SL7207(PVAXD)、PBS间均存在统计学差异(P<0.01),并在第6周达到最高值;中和实验显示该疫苗诱导的血清IgG具有中和活性。结论 证实以减毒沙门菌为载体的TGEV S基因疫苗口服免疫仔猪有较好的免疫原性。     

Transplacental transfer of schistosomal circulating anodic antigens in cows

The present work investigated the transplacental passage of circulating anodic schistosome antigens (CAA) and the production of foetal antibodies in response to antigenic stimulation in Schistosoma mattheei infected cows. Three groups were available: six calves born to non-infected cows received colostrum from a pool from non-infected cows (group 1), six calves born to non-infected cows (group 2) and six calves born to infected cows (group 3) received colostrum from a pool from infected cows. Schistosoma-specific IgG1 antibody and CAA levels were measured in the colostrum pools, the sera collected from the cows, and the sera collected from the calves at birth, after intake of colostrum and at day 30. The specific IgG1 antibody levels were significantly higher in the sera from cows of group 3. In four cows of group 3 high CAA levels were detected. The specific IgG1 antibody levels were 0.646 and 0.176 OD for the infected and non-infected colostrum pool, respectively, and the CAA levels were 5667 and 2557 pg CAA/mL, respectively. At birth high levels of specific IgG1 antibody and CAA were detected in 4 calves of group 3; levels in the other two calves were negligible. After intake of colostrum, specific IgG1 antibody levels of group 1 increased slightly at day 1 to become again insignificant at day 30. In group 2 specific IgG1 antibody levels increased significantly between days 0 and 1, to decrease, although not significantly, at day 30. Finally, in group 3 the delta OD values increased at day 1 and remained high until day 30. After intake of colostrum the CAA level increased very slightly for groups 1 and 2 to become again undetectable at day 30. In group 3 a nonsignificant decrease in CAA levels was observed at day 1 followed by a further significant decrease to reach low levels at day 30. The suggested intrauterine antigenic stimulation may be important not only for generating immune responses to natural early infections, but also for enhancing the immunogenicity and efficacy of vaccines administered to newborns.     

Prevention of human rotavirus infection with chicken egg yolk immunoglobulins containing rotavirus antibody in cat

A study was made on the passive protection against rotavirus-induced diarrhea. Chickens were immunized with bovine rotavirus (serotype 1) and the egg yolk immunoglobulins containing a high titer anti-rotavirus neutralizing antibody (CEYI) was obtained. The CEYI was then orally administered to specific-pathogen-free cats, and the cats were infected with human rotavirus. The cats treated with the CEYI remained clinically healthy after challenge, whereas diarrhea occurred in the placebo-fed cats as control. Virus antigens were detected in feces in all the diarrheal cases in the placebo-fed cats but were only sporadically detected in the CEYI-fed cats. However, the cats were only protected against rotavirus infection by the presence in the gut at the time of infection of the antibody. These results suggested that continuous administration of the CEYI is capable of preventing children from diarrhea induced by human rotavirus infection and viral shedding.     

Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum

Cryptosporidiosis is a zoonotic protozoan disease that affects the gastrointestinal tract of animals and humans. Diarrhoea as the most important indication of the infection leads to high economic losses in livestock industries and is a life threatening infection in immunocompromised individuals. In the absence of the effective drugs, vaccine has an effective role in the prevention of infection. For this purpose we developed a vaccine utilizing recombinant P23 protein and immunized pregnant cows four times from 70 days to parturition every 2 weeks. After parturition, each calf received his dam colostrum and challenged with 1 × 10⁷ Cryptosporidium parvum oocysts at 12 h of age. Results showed that in contrast with the control group, the antibody titre in the sera and first milking colostra of the immunized cows significantly increased and calves fed hyperimmune colostrum did not show cryptosporidiosis signs. Moreover, enriched colostrum not only reduced significantly the amount of oocyst excretion but also delayed its onset. Our study showed that recombinant P23 protein could be used for passive immunization of newborn calves against Cryptosporidium parvum.     

Maternal antibody-mediated protection against gastroenteritis due to rotavirus in newborn mice is dependent on both serotype and titer of antibody

In order to evaluate the role of passively acquired, rotavirus-specific antibodies in protection against diarrhea, we inoculated mouse dams with rotaviruses of various serotypes, and their newborns were orally challenged with a primate rotavirus (simian SA-11). Dams were immunized by using a regimen that included repeated inoculations administered either orally or intraperitoneally with adjuvant. The serum antibody response detected in dams by radioimmunoassay and plaque-reduction neutralization after parenteral immunization was approximately 15-fold and 80-fold greater, respectively, than that found after oral "hyperimmunization." Parenteral immunization with rotavirus serotypes either homotypic or heterotypic to the challenge virus protected suckling mice against diarrhea; protection was closely correlated with the in vitro neutralizing activity of maternal serum against the challenge virus. Oral immunization with only rotavirus strains homotypic to the challenge virus afforded protection; the lower immune response after oral immunization with rotaviruses heterotypic to the challenge virus resulted in a titer of neutralizing antibody to the challenge virus below the protective threshold. From our current studies it appears that antibody-mediated passive protection against rotavirus challenge is dependent on both serotype and titer of antibody.     

Acquired resistance to Fasciola hepatica in cattle using a purified adult worm antigen

Calves were immunized twice in 4 weeks with a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen (denoted FhSmIII(M)) isolated from F. hepatica adult worm extracts by antibody affinity chromatography and challenged 7 weeks later with F. hepatica metacercariae. Flukes were recovered at 16 weeks of infection at which time the immunized calves had 55% less F. hepatica than the controls. All of the immunized calves developed high antibody levels of FhSmIII(M), detectable in the ELISA, by 4 weeks after a single immunization. By 9 weeks of infection with F. hepatica the immunized calves had lower sorbitol dehydrogenase levels than the unimmunized, F. hepatica-infected control calves, indicating less liver damage in the vaccinated group. These studies demonstrate that subcellular F. hepatica macromolecules cross-reactive and cross-protective against S. mansoni also have the potential to serve as vaccines in cattle exposed to this parasitic disease.     

旋毛虫在小鼠先天性传播的初步研究

目的?摇研究旋毛虫在小鼠的先天性传播并观察母鼠抗旋毛虫抗体对攻击感染的保护作用。 方法 将昆明小鼠分为受孕后感染组和感染后受孕组，子鼠出生后1 d内剖杀，检查旋毛虫幼虫；将正常母鼠所产子鼠由感染旋毛虫的母鼠喂养，21 d后宰杀，检查旋毛虫幼虫。用间接ELISA检测感染母鼠所产子鼠出生后不同时间的血清抗旋毛虫抗体，观察母鼠抗旋毛虫抗体对攻击感染的免疫保护。 结果 受孕后7 d感染旋毛虫的母鼠所产的6只子鼠中有2只感染旋毛虫；感染旋毛虫后8 d和22 d受孕雌鼠所产子鼠的感染率分别为20%（2/10）和25%(2/8)，从子鼠检获的旋毛虫均是未成囊的幼虫。交叉哺乳实验表明正常母鼠所产的30只子鼠未见旋毛虫感染。感染母鼠所产27只子鼠出生后1、7、24及40 d的血清抗体阳性率分别为100%、100%、77.8%及14.8%，子鼠出生后40 d攻击感染的减虫率为62.0%；感染母鼠所产子鼠血清被动转移小鼠的减虫率55.7%，与对照组比较差异有显著性（P<0.01）。 结论 旋毛虫在小鼠可经胎盘传播，母鼠的抗旋毛虫抗体对子鼠抗攻击感染可能具有部分保护作用。     

Serum antibody as a marker of protection against natural rotavirus infection and disease

To determine whether naturally acquired serum IgA and IgG antibodies were associated with protection against rotavirus infection and illness, a cohort of 200 Mexican infants was monitored weekly for rotavirus excretion and diarrhea from birth to age 2 years. Serum samples collected during the first week after birth and every 4 months were tested for anti-rotavirus IgA and IgG. Children with an IgA titer >1:800 had a lower risk of rotavirus infection (adjusted relative risk [aRR], 0.21; P<.001) and diarrhea (aRR, 0. 16; P=.01) and were protected completely against moderate-to-severe diarrhea. However, children with an IgG titer >1:6400 were protected against rotavirus infection (aRR, 0.51; P<.001) but not against rotavirus diarrhea. Protective antibody titers were achieved after 2 consecutive symptomatic or asymptomatic rotavirus infections. These findings indicate that serum anti-rotavirus antibody, especially IgA, was a marker of protection against rotavirus infection and moderate-to-severe diarrhea.     

Virus-specific IgM antibodies in acute gastroenteritis due to a reovirus-like agent (rotavirus).

62 serum samples from 24 patients with rotavirus gastroenteritis were tested for IgM antibodies against a bovine rotavirus by an indirect fluorescent antibody technique. IgM antibodies were detected in one or more of the serum samples from all but one of the patients. IgM antibodies were not detected in samples obtained from 11 of the patients after the 5th week of illness. Absorption of sera for IgG with Staphylococcus aureus increased the sensitivity of the IgM antibody test. It is concluded that the presence of IgM antibodies against bovine rotavirus in a patient's serum, as measured by the present technique, does suggest a recent rotavirus infection. On the other hand, the lack of IgM antibodies in the serum of a child with acute gastroenteritis between the second and the 5th week of illness tends to exclude rotavirus as a cause of the disease.     

Vaccination of dogs against Babesia canis infection using parasite antigens from in vitro culture

Groups of five dogs were vaccinated with different Babesia canis vaccine formulations. It appeared that partial protection against challenge infection was obtained when using parasite antigens from in vitro culture in combination with saponin. Protection was evident as a decrease in parasitaemia after challenge and was associated with the presence of serum antibodies against Babesia parasites. In addition, parasite antigen derived from in vitro culture supernatant exhibited more protective activity than somatic parasite antigen, in that a less marked fall of haematocrit values was found after challenge infection. The fall of haematocrit value observed in the animals immunized with somatic parasite antigen was not different from that observed in the adjuvant control group.     

Immunoglobulin components and anti-viral activities in bovine colostrum

Bovine colostrum whey and immunoglobulins were prepared. Their characteristics and anti-viral activities were studied:IgG, IgA and IgM were found in bovine colostrum. Most IgG was polymerized. Although neutralization activities against bovine, simian and human rotaviruses existed, anti-human adenovirus antibody was not found. Effects on prophylaxis and treatment for rotavirus gastroenteritis were expected.