猪霍乱沙门菌菌蜕的制备及免疫保护效力研究

为制备猪霍乱沙门菌菌蜕并研究其免疫保护效力,克隆噬菌体phi X174裂解基因E,与p BV220连接,构建温控溶菌表达载体,将该载体转入猪霍乱沙门菌TTB1中,制备菌蜕并对其裂解率、安全性以及对小鼠的免疫保护效力进行了研究。结果显示:成功克隆了裂解基因E、片段大小274 bp,构建了温控溶菌质粒载体p BVE,制备了猪霍乱沙门菌菌蜕,其裂解率最高为99.46%、经冻干灭活残余活菌,安全性试验检测证实其安全后,对小鼠进行了免疫保护试验,其保护率为70%、与弗氏佐剂灭活苗保护率相当、优于甲醛灭活苗。研究为猪霍乱沙门菌感染的防治奠定了基础。     

猪源致病性大肠杆菌菌蜕的制备

为制备仔猪水肿病源致病性大肠杆菌菌蜕,本实验克隆phiX174裂解基因E,构建温控溶菌表达载体,将其转入猪源致病性大肠杆菌JLBC1501中,制备菌蜕计算其裂解率,并用电镜进行观察。结果显示:扩增出片段大小为274bp的裂解基因E,成功构建温控溶菌质粒载体pBVE,制备了猪源致病性大肠杆菌菌蜕,其裂解率最高为99.72%,扫描电镜观察,菌蜕株内容物大量排出、细菌发生明显皱缩。研究结果为仔猪水肿病的防治奠定了基础。     

猪致病性大肠杆菌O9菌蜕的制备及免疫保护力

通过PCR方法克隆噬菌体PhiXl74的融菌基因E,将其与温控表达载体pBV220连接,成功构建温控融菌表达盒,设计扩增温控表达盒的2对引物,使其5′端分别与lacZ基因敲除基因的首尾50bp基因同源,使用Red系统同源重组,使用麦康凯平板筛选、PCR鉴定白色菌落,温控诱导融菌蛋白的表达,制备大肠杆菌O9的菌影,并对其融菌效率及免疫保护力进行了初步研究。结果显示,成功构建了温控融菌表达盒,经同源重组成功获得猪致病性大肠杆菌O9温控融菌株,温控表达溶菌蛋白能够成功抑制细菌的增殖,并使活菌数下降3个指数,对小鼠安全性好,皮下免疫组攻毒保护率达到71.43%。     

鸭源大肠杆菌O_(78)菌蜕的制备及免疫原性研究

为制备大肠杆菌(E.coli)菌蜕并研究其免疫原性,本研究将带有裂解基因E的质粒pHH43转化致病性鸭源E.coli O78,构建获得E.coli O78/pHH43重组菌。于不同培养温度下研究重组菌E裂解蛋白表达所介导的宿主菌的裂解情况,结果显示在42℃培养时最佳诱导时间为4h,约90%的重组菌被裂解失活,以冻干法及添加庆大霉素完全灭活未裂解的细菌后制备"菌蜕疫苗"。雏鸭免疫试验表明,接种E.coli O78/pHH43菌蜕两周后免疫雏鸭可产生对同源强毒菌株致死剂量攻击的免疫保护。比较E.coli O78/pHH43菌蜕与甲醛灭活的同株E.coli所诱导的免疫保护效果,结果免疫保护率分别为87.5%和75%,表明菌蜕比甲醛灭活法能更有效地诱导机体的特异性免疫应答。     

鸡白痢沙门菌pagC基因缺失株菌蜕疫苗的制备和免疫保护效果评价

为了在鸡白痢沙门菌pagC基因缺失突变株的基础上制备可用于鉴别诊断的菌蜕疫苗并评价其免疫保护效果,本研究利用细胞穿膜肽MAP制备ΔPagC菌蜕,透射电镜和荧光显微镜观察菌蜕形态和核酸残留情况;将ΔPagC菌蜕与佐剂ISA206混合乳化制备菌蜕疫苗并进行无菌检测;基于ICR小鼠评价菌蜕疫苗对鸡白痢沙门菌CVCC1800、肠炎沙门菌ATCC19585和鼠伤寒沙门菌CVCC542感染的交叉保护效果;结合基于PagC蛋白的沙门菌血清学检测方法评价pagC基因缺失株菌蜕疫苗的鉴别诊断效果。结果显示,菌蜕制备最佳条件为用终浓度为50μmol/L MAP溶液处理OD_{600 nm}=0.3的菌液1 h以上,灭活效果可达99.00%;菌蜕保持基本细胞形态的同时内容物大量释出,核酸残留水平较低;菌蜕疫苗针对上述3种血清型沙门菌的免疫保护率分别为25%、100%和100%;菌蜕疫苗与基于PagC蛋白的沙门菌血清学检测方法联用可在一定程度上起到鉴别诊断作用。结果表明,制备所得菌蜕疫苗在保护小鼠的同时,能够在一定程度上区分人工免疫和自然感染,为沙门菌的净化工作提供帮助。     

利用温控双表达载体制备印第安纳沙门菌菌蜕

为制备安全高效的印第安纳沙门菌菌蜕,本研究以温控质粒pTCV为载体,分别构建出只含噬菌体PhiX174裂解基因E的pTCK01质粒、同时含裂解基因E和金黄色葡萄球菌核酸酶A(SNA)的pTCK02质粒及对裂解基因E和SNA密码子优化后的p TCK03质粒,分别转化印第安纳沙门菌S1105后经42℃诱导表达,并对各重组菌开展溶菌动力学监测、重组菌的基因组电泳检测和菌蜕电镜观察。PCR鉴定结果表明,重组质粒pTCK01、pTCK02和pTCK03均正确构建。溶菌动力学显示,含质粒pTCK03的重组菌S1105(pTCK03/S1105)的OD_(600nm)值在诱导后120 min达到最低,且低于重组菌p TCK01/S1105和重组菌pTCK02/S1105,之后一直稳定在最低水平;其溶菌率也显著高于pTCK01/S1105和p TCK02/S1105(p0.05)。基因组电泳检测结果显示,pTCK03/S1105基因组在诱导1 h后即被降解且降解得最彻底。扫描和透射电镜观察结果显示,pTCK03/S1105菌蜕表面形态明显皱缩,胞质内容物完全释放。上述结果表明,通过优化裂解基因E和SNA密码子可提高印第安纳沙门菌菌蜕的裂解效率,并彻底降解了其遗传物质,本研究为印第安纳沙门菌菌蜕疫苗的研发提供新思路和参考。     

犬布鲁氏菌菌壳疫苗的制备简

菌壳技术是一种新型的灭活疫苗制备方法,通过非变性的灭活方式保存细菌表面多个抗原表位,所制备的菌壳可作为预防细菌病的理想疫苗。本试验从噬菌体PhiX174 DNA钓取裂解E基因,连接至温控原核表达载体pBV220,通过PCR在所构建的pBV220+E上扩增出蛋白裂解部件（protein lysis component,PLC）,该部件包含阻遏蛋白cI857、溶菌E基因及终止序列rrnbT1T2,然后将其克隆至广宿主表达载体pBBR1MCS-2中,最终将构建的广宿主裂解质粒pBBR+PLC电转入犬布鲁氏菌RM6/66中。试验结果表明,经42 ℃诱导后,广宿主裂解质粒对犬布鲁氏菌RM6/66的裂解率达100%,成功制备了犬布鲁氏菌RM6/66菌壳疫苗。本试验通过菌壳技术制备的犬布鲁氏菌疫苗对预防宠物犬布鲁氏菌病起到重要作用,同时也对人兽布鲁氏菌疫苗的研制提供新策略。     

粗糙型布鲁菌M111菌壳的制备

将含有裂解酶基因重组温控裂解质粒pBBR1MCS：：PR-PL-E电转化至粗糙型布鲁菌M111中，构建重组布鲁菌M111（pBBRlMCS：：PR-PL-E）。重组菌株在28℃培养，42℃诱导表达裂解酶E，从而制备布鲁菌菌壳。绘制布鲁菌生长曲线及裂解曲线，计算裂解率并用透射电镜观察布鲁菌菌壳的形态。结果显示，成功制备了布鲁菌菌壳，温控裂解质粒pBBRIMCS：：PR-PL-E对布鲁菌的裂解率为100％。透射电镜观察可见细菌内容物部分流出，细菌表面出现不同程度的皱缩，细胞形态发生变化。结果表明，本试验成功制备了粗糙型布鲁菌菌壳，初步研究了其基本特性，为下-步开展布鲁菌菌壳疫苗的研究奠定了基础。     

禽致病性大肠杆菌(APEC)菌蜕的制备研究

菌蜕是通过PhiX174噬菌体溶菌基因E的可调控表达而形成的、缺少细胞浆和核酸且无繁殖能力的革兰氏阴性细菌菌体。这种非变性的灭活方式使菌蜕完好保留了细菌的各种抗原成分和免疫黏附分子,具有优良的免疫原性和固有的免疫佐剂性质以及免疫系统靶向性质。菌蜕的这些生物学特点使其在作为新型非变性灭活菌苗、作为递呈异源抗原的重组疫苗以及作为DNA疫苗甚至药物的载体方面都显现出巨大的研究开发前景。本研究成功构建了热敏诱导、pR、pL串联双启动子表达E基因的、具有不同抗性标记和复制起点的系列溶菌载体,比国外用pR或pL单启动子的溶茵载体在K-12株大肠杆菌(如XL1-blue)的溶茵效率高一个指数级。本研究还成功制备出禽致病性大肠杆菌(Avian pathogenic E.coli,APEC)野毒株的菌蜕,为进一步进行菌蜕疫苗的开发研究奠定了技术基础。     

粗糙型布鲁菌M111菌壳的制备

将含有裂解酶基因重组温控裂解质粒pBBR1MCS∷PR-PL-E电转化至粗糙型布鲁菌M111中,构建重组布鲁菌M111(pBBR1MCS∷PR-PL-E)。重组菌株在28℃培养,42℃诱导表达裂解酶E,从而制备布鲁菌菌壳。绘制布鲁菌生长曲线及裂解曲线,计算裂解率并用透射电镜观察布鲁菌菌壳的形态。结果显示,成功制备了布鲁菌菌壳,温控裂解质粒pBBR1MCS∷PR-PL-E对布鲁菌的裂解率为100%。透射电镜观察可见细菌内容物部分流出,细菌表面出现不同程度的皱缩,细胞形态发生变化。结果表明,本试验成功制备了粗糙型布鲁菌菌壳,初步研究了其基本特性,为下一步开展布鲁菌菌壳疫苗的研究奠定了基础。     

Evaluation of a live Salmonella choleraesuis vaccine by intranasal challenge.

Weaned pigs were immunised with a live attenuated Salmonella choleraesuis vaccine. The protective immunity induced was compared with that of unvaccinated pigs by intranasal challenge with a field strain of S choleraesuis. Of 18 unvaccinated pigs, three died as a result of challenge from S choleraesuis and 10 of the survivors developed chronic lung lesions. None of the vaccinated pigs died as a result of challenge and two only showed macroscopic evidence of salmonellosis on autopsy three weeks after challenge. It was concluded that subcutaneous vaccination of pigs prevented death and clinical illness but did not prevent invasion of tissues following intranasal challenge with S choleraesuis.     

Plasmid profile analysis, phage typing, and antibiotic sensitivity of Salmonella dublin from clinical isolates in the United States.

One hundred clinical isolates of Salmonella choleraesuis subsp. choleraesuis serovar dublin (Salmonella dublin) were examined for phage sensitivity, antibiotic resistance patterns, and plasmid content. Computer analysis of the lysis patterns observed by using 27 typing phages divided the S. dublin isolates into 26 groups. One lytic pattern (Designated pattern 16) contained 52% of the isolates examined whereas 16 isolates had unique patterns, and nine patterns had fewer than ten members. Although 14 antibiotic resistance patterns were observed among the 100 isolates, 79% of the isolates grouped in three major patterns. Seven plasmid groups were identified and designated A-G based on the large plasmids found in the isolates. Of the 100 isolates, 28 contained the plasmid profile of Group A, 28 were Group B, 7 were Group C, 34 were Group D, and 1 isolate each was observed in Groups E, F, and G. The strong association between antibiotic resistance pattern and plasmid type suggest that the drug resistance genes are plasmid borne.     

猪丹毒丝菌CbpB蛋白对小鼠的免疫效果分析

探究猪丹毒丝菌（ER）CbpB蛋白的免疫原性和保护作用,为深入研制ER基因工程亚单位疫苗提供新的思路和技术储备。以表达的ER重组蛋白CbpB、SpaA、CbpB+SpaA以及ER灭活全菌体（V-AEr21）、商品化弱毒疫苗、商品化灭活疫苗分别免疫小鼠。利用间接ELISA检测小鼠血清中IgG抗体,血清杀菌试验测定功能性抗体水平,攻毒试验测定免疫保护率,组织荷菌数试验测定ER定植情况,并采集小鼠组织脏器（脾、肺、肝、肾）制备切片,观察病理变化。结果显示：与免疫原性最优的商品化弱毒疫苗相比,CbpB和SpaA虽产生的IgG抗体均仅为1∶6 400,但血清杀菌效果强;对小鼠的免疫攻毒保护率与商品化弱毒疫苗相同,均为100%,且在脾、肺、肝、肾组织中均无细菌定植,病理组织学观察与空白对照无明显区别。CbpB重组蛋白具有良好的免疫原性和保护作用,能刺激机体产生体液免疫和细胞免疫,可以作为猪丹毒基因工程亚单位疫苗的候选抗原。     

鸭疫里默氏杆菌多价灭活苗研制的研究报告

用三个血清型的菌株,分别是JYL-1、JYL-2、JYL-7,用两种佐剂,分别是蜂胶和油乳剂共制备了两种多价菌苗,即JYL-1、JYL-2、JYL-7（1、2、7三个血清型）三个RA血清型混合多价蜂胶苗和油乳剂苗。试验结果表明。蜂胶复合佐剂多价苗具有产生免疫保护速度快,免疫持续时间长的优点,接种后第3天即产生部分免疫保护力,第120天时仍具有完全保护力;油乳剂多价苗产生保护力的速度较慢,接种后第10天时开始表现出部分免疫保护,其完全保护力也可持续到接种后第120天。免疫后13d时,免疫保护率可达90％左右。免疫后16d时保护率为100％。免疫后120d时,两种多价菌苗的免疫保护率均可达到100％,免疫后150～180d时,免疫保护率可达800左右。统计学分析证明,菌苗的安全性良好。用3个免疫剂量的菌苗所做的安全试验表明无明显不良反应,菌苗在4℃保存一年,18～22℃室温保存4个月不影响菌苗的免疫效力。田间试验表明,用菌苗免疫鸭群后,可有效地使鸭群抵抗鸭疫里默氏杆菌的感染。     

Immunization of mice and calves against Salmonella dublin with attenuated live and inactivated vaccines.

Previous findings, viz. that mice can be successfully immunized against infection with Salmonella dublin with either live or inactivated vaccine, were confirmed. Immunity lasted for at least 12 weeks in mice which had been immunized with inactivated alum-precipitated vaccine. The immunogenicity of inactivated vaccine gradually decreased on storage at 4 degrees C, but this was only detectable if a single injection was used for immunization: 2 injections virtually eliminated this phenomenon. The immunogenicity of live vaccine in mice was not enhanced by levamizole or the simultaneous injection of inactivated organisms. Both live and inactivated vaccines provided immunity in calves. A single injection of lyophilized vaccine, prepared from live rough Salmonella dublin strain (HB 1/17),protected 3 out of 6 calves, while 2 injections of a formalin-inactivated, alum-precipated vaccine, containing 1% packed cells of S. dublin strain 2652 V, protected 5 out of 6 calves against intraduodenal challenge with 2 x 10(9), S. dublin strain 2652 V. Two calves which had been immunized with an inactivated oil adjuvant vaccine were also solidly immune to this challenge. Serum antibody response in calves was poor when measured by the tube agglutination and the haemagglutination tests. Similarly, the sera had only marginal protective values when tested by means of a passive protection test in mice. Antibody titres alone are not a valid measure therefore, for the immune status of immunized animals.     

鸭疫里默氏杆菌贵州流行株蜂胶灭活疫苗制备

【目的】制备鸭疫里默氏杆菌(Riemerella anatipestifer,RA)贵州流行株蜂胶灭活疫苗。【方法】选取RA血清2型贵州流行株(RA-SS-8株)为基础菌株,依次利用分光光度法和平板计数法测定细菌生长曲线,再进行灭活条件筛选,以蜂胶为佐剂制备RA贵州流行株蜂胶灭活疫苗,并进行无菌检验、安全性检验及免疫鸭攻毒保护性试验。【结果】分光光度法测定RA-SS-8株菌液D_{600 nm}值随培养时间变化显示,细菌在0~3 h时增殖缓慢,在3~10 h时增殖趋势明显加快,在10 h以后增殖逐渐趋于平缓,随着培养时间的延长,细菌最终进入衰亡期;平板计数法结果显示,RA-SS-8株D_{600 nm}值为0.1~0.8时,该菌处于对数生长期,且D_{600 nm}值与活菌数呈现良好的线性关系;RA-SS-8株最佳灭活条件为0.2%甲醛溶液、37 ℃灭活12 h;蜂胶灭活疫苗含菌量为3.8×10⁹ CFU/mL,蜂胶干物质含量为10 mg/mL;无菌检验巧克力琼脂培养基上未见菌落生长;安全性检验以2倍免疫剂量接种雏鸭在观察期内未表现出不良反应,大体病变观察未见明显病变;免疫鸭攻毒保护性试验显示,蜂胶灭活疫苗免疫组鸭对RA-SS-8株的攻击后保护率为70%,蜂胶灭活疫苗对试验鸭心脏、肝脏组织均具有良好的保护效果。【结论】本研究成功制备了RA贵州流行株蜂胶灭活疫苗,为蜂胶灭活疫苗制备和动物免疫试验奠定基础。