产气荚膜梭菌α毒素基因的克隆、表达及其抗血清的制备

本试验通过设计并合成1对引物,PCR扩增B型产气荚膜梭菌C58-2株α毒素完整成熟肽序列,并将其插入到pGEM-T Easy载体中,构建克隆载体pGEM-T-α。对克隆载体pGEM-T-α进行EcoRⅠ和Hind Ⅲ的双酶切,将得到的1125 bp片段以正确的阅读框架定向克隆于pET-28a(+)中,然后将重组质粒转化大肠杆菌BL21(DE3)plys宿主菌中,37 ℃、1.0 mmol/L IPTG诱导该片段获得良好表达。经SDS-PAGE分析,其表达的蛋白约为46.1 ku,与预期大小一致。Western blotting结果显示,该重组α毒素蛋白可被A型产气荚膜梭菌定型血清识别,表明该重组α毒素蛋白具备与天然毒素相似的反应原性。重组α毒素蛋白在菌液上清、超声波裂解上清和超声波裂解沉淀中均有分布,且以包涵体表达为主,表明重组α毒素蛋白可同时在胞外、周质和胞浆表达。小鼠毒力试验结果表明,重组α毒素蛋白不具有毒性。毒素—抗毒素中和试验结果表明,该抗血清具有α毒素特异性。以重组α毒素蛋白作为抗原免疫家兔制备血清,效价测定结果表明每1 mL重组α毒素蛋白抗血清可以中和100 MLD的A型毒素。     

产气荚膜梭菌β毒素的表达及其抗血清的制备

PCR扩增B型产气荚膜梭菌C58-2株β毒素完整成熟肽序列,将942 bp的基因片段以正确的阅读框架定向克隆于p ET-32a(+)中,然后将重组质粒转化进宿主菌BL21(DE3)中,在37℃1 mmol/L IPTG诱导下该片段获得良好表达。经SDS-PAGE分析,其表达的蛋白约为54.6 ku,与预期值一致。Western blot显示,该重组蛋白可被C型产气荚膜梭菌血清识别,表明该重组蛋白具备与天然毒素相类似的反应原性。重组β毒素蛋白在菌液上清、超声波裂解上清和包涵体中均有分布,且以包涵体为主,表明重组蛋白可同时以胞外、周质和胞浆的形式表达。小鼠试验表明,重组β毒素蛋白不具有毒性。毒素-抗毒素中和试验表明,该抗血清具有β毒素特异性。以重组β毒素蛋白作为抗原免疫家兔,每0.1 m L的一免、二免、三免后抗血清分别可以中和10、20、50个小鼠MLD的C型毒素。结果表明,试验所获得的重组菌株有望成为产气荚膜梭菌β类毒素生产的候选菌株,所制备的抗血清有望进一步研制成为抗β毒素国家标准品。     

产气荚膜梭菌ε毒素原核表达及其单克隆抗体的制备

以大肠杆菌表达的D型产气荚膜梭菌点突变的ε毒素蛋白作为免疫原,免疫BALB/c小鼠制备单克隆抗体,经过ELISA方法筛选,获得2株稳定分泌抗ε毒素单克隆抗体的杂交瘤细胞株2C3-D7和5H6-B8,单抗均为IgG1型。Western blot鉴定结果显示:2株单抗均能与重组ε毒素蛋白及D型产气荚膜梭菌ε毒素蛋白发生特异性结合。本试验为进一步研究产气荚膜梭菌ε毒素的生物学特性及建立产气荚膜梭菌的快速检测方法提供了基础。     

产气荚膜梭菌ε毒素及其疫苗研究进展

产气荚膜梭菌病是由产气荚膜梭菌引起的一种重要的人兽共患传染病,可导致山羊、绵羊等动物的肠毒血症或坏死性肠炎,并且可引起动物脑、心、肺和肾组织的水肿.B型和D 型产气荚膜梭菌所产生的ε毒素是引起动物上述病理变化和死亡的重要因素之一.虽然甲醛灭活的毒素疫苗能对动物产生保护性抗体,但是,灭活苗潜在的安全性原因使其在应用上受到限制.因此,基于ε毒素基因的重组疫苗和弱毒疫苗就成为人们研究的目标.     

D型产气荚膜梭菌α毒素基因的克隆与高效表达

为建立产气荚膜梭菌(Clostridium perfringens)α毒素(cpa)克隆表达方法,应用PCR方法从羊源D型产气荚膜梭菌中扩增cpa成熟肽基因序列,将其插入pET-28b载体中,构建重组表达载体pET-28b-cpa;通过PCR扩增、双酶切和测序方法对重组载体进行鉴定及序列分析,然后转入BL21(DE3)pLysS中诱导表达;用SDS-PAGE检测目的蛋白大小及分布,Western blotting方法检测其反应原性。结果表明,所扩增的cpa基因大小为1110 bp,与GenBank参考序列同源性为99%以上;SDS-PAGE分析结果表明,目的蛋白大小为41.2 ku,与预期大小一致,在超声波裂解上清和包涵体中均有分布,但主要以包涵体为主,两者分布均具有和天然毒素相似的反应原性。     

产气荚膜梭菌ε毒素研究进展

产气荚膜梭菌是一种重要的人畜共患细菌性传染病病原,在自然界中分布极广,一定条件下可引起多种疾病.该菌常通过其外毒素危害人和动物机体,所产毒素主要有α、β、ε和ι4种外毒素.根据毒素产生的情况,将产气荚膜梭菌分为A、B、C、D、E、F和G7个型.ε毒素毒力最强,主要由B型和D型菌分泌.论文通过致病机理、病理变化、流行特点...     

产气荚膜梭菌β1毒素基因克隆与表达

以C型产气荚膜梭菌中PCR扩增出β1毒素基因,构建了表达质粒p GEXKG-β1,将构建的KG-β1转化受体菌BL21(DE3),得到重组菌株BL21/KG-β1。对重组菌株诱导的表达产物进行了SDS-PAGE分析,结果表明重组菌株可以高效表达毒素蛋白。     

产气荚膜梭菌ε毒素重组突变体大肠杆菌表达条件的优化

为优化产气荚膜梭菌ε毒素重组蛋白的表达条件,以前期构建的产气荚膜梭菌ε毒素重组突变体为基础,通过控制变量法确定诱导温度、诱导时间、IPTG诱导浓度以及诱导时菌液浓度等原核蛋白表达条件.结果表明,ε 毒素的重组突变体在37℃、菌液OD600值为0.895～1.295,1.6 mM IPTG诱导6 h条件下可溶性表达量最高...     

产气荚膜梭菌ε毒素突变体的表达及免疫保护力评价

为获得产气荚膜梭菌ε毒素(ETX)的重组突变体,并评价其毒力及免疫原性。对已知的D型产气荚膜梭菌ETX编码基因进行优化设计,并将第106位组氨酸突变为脯氨酸,经人工合成获得基因片段GETXH106P。将GETXH106P克隆至原核表达载体p ET30a(+),转染BL21(DE3)菌中进行表达与纯化,从而获得重组蛋白r ETXH106P。利用Western blot方法检测r ETXH106P与D型产气荚膜梭菌抗血清的反应性,并检测其对犬肾细胞系(MDCK细胞)以及小鼠的毒力。随后,将r ETXH106P与Montanide ISA 201佐剂混合乳化制备疫苗,免疫4只健康家兔,按照《中华人民共和国兽药典》(2015年版)规定的方法检测一免及二免后兔血清的中和抗体效价。结果表明,r ETXH106P为可溶性表达,通过灰度扫描,其可溶性表达量比例可达30%;该重组蛋白能与D型产气荚膜梭菌毒素抗血清反应;细胞毒性试验结果显示,100μg/m L的重组蛋白仍无细胞毒性;小鼠安全性实验结果显示,6.25×106ng/kg剂量的重组蛋白对小鼠仍无致死性;每毫升的一免抗血清可中和450~700个最小致死量(MLD)、二免抗血清可中和3000~4000个MLD的D型产气荚膜梭菌毒素;用1 MLD的D型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔4/4保护。以上结果表明,产气荚膜梭菌r ETXH106P重组蛋白毒力基本消失,且保留了良好的免疫原性,是D型产气荚膜梭菌病基因工程亚单位疫苗的理想候选抗原蛋白。     

D型产气荚膜梭菌ε毒素研究进展

D型产气荚膜梭菌常引起新生羔羊痢疾和山羊、牛等家畜的肠毒血症,致死性强,对畜牧业造成很大的危害.ε毒素是D型产气荚膜梭菌的主要致死性毒力因子和保护性抗原,从分子生物学的角度对该毒素的研究具有重要实际意义.论文主要对国内外关于D型产气荚膜梭菌ε毒素分子生物学结构与性质,ε毒素引起的家畜肠毒血症以及对其作用靶器官肾脏和脑组织破坏的致病机理进行了介绍,对国内外D型产气荚膜梭菌ε毒素预防措施进行了综述,为研究D型产气荚膜梭菌ε毒素结构与功能的关系以及预防治疗动物肠毒血症提供参考.     

产气荚膜梭菌ε毒素蛋白高效表达体系的构建及其免疫保护性的研究

ε毒素是由B和D型产气荚膜梭菌产生的,能够引起牛、羊肠毒血症,给畜牧业造成巨大的经济损失。本试验将ε毒素基因片段(972 bp)以正确的阅读框架定向连接到pET-28a(+)质粒上,然后将重组质粒导入受体菌株,成功构建重组菌株BL21(DE3)。SDS-PAGE和Western blotting检测结果表明,重组的菌株BL21(DE3)能以包涵体的形式高效表达ε毒素(占菌体总蛋白的32.83%)。在此基础上,用有效剂量0.2 mg包涵体粗提物免疫家兔,抗血清间接ELISA检测结果显示,在第4周抗体效价达到最高值为8.7log2。因此,在本研究中构建的重组菌株BL21(DE3)能高效表达ε毒素,而且该毒素具有较高的免疫保护性。     

C、D型二价产气荚膜梭菌抗毒素血清的制备及保护效果评价

为治疗产气荚膜梭菌感染引起的疾病,研究制备了产气荚膜梭菌多价高效抗毒素血清。试验采用C、D型产气荚膜梭菌标准菌株,制备了高浓度外毒素和灭活疫苗,作为免疫原多次免疫绵羊,通过间接ELISA法监测绵羊抗体水平变化,采用小鼠中和试验检验绵羊抗毒素血清保护效果。结果表明:制备的高效价抗C、D型产气荚膜梭菌毒素血清每0.1 m L血清能中和400个C型毒素对小鼠的MLD和600个D型毒素MLD,有良好的应用前景。     

产气荚膜梭菌主要外毒素最新研究进展

产气荚膜梭菌是一种重要的人兽共患病的病原体,可引起人的食物中毒和抗生素相关腹泻.近年来,对产气荚膜梭菌的主要外毒素研究取得了重大进展.本文综述了产气荚膜梭菌主要外毒素的组成、结构、理化性质、作用机理、检测及分子遗传学等方面的研究进展.     

Recombinant unpurified rETXH106P/CTB-rETXY196E protects rabbits against Clostridium perfringens epsilon toxin

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, has been touted as a potential biological weapon and is known to induce fatal enterotoxemia in a variety of livestock animals. For the efficient production of recombinant proteins with the objective of investigating the effects of different recombinant vaccines against ETX, a bicistronic design (BCD) expression system including the ETX coding sequence with mutation of amino acid 106 from Histidine to Proline (ETX^H106P) in the first cistron, followed by Cholera Toxin B (CTB) linked with the ETX coding sequence with mutation of amino acid 196 from Tyrosine to Glutamic acid (ETX^Y196E) in the second cistron, was generated under the control of a single promoter. Rabbits were immunized twice with five inactivated recombinant Escherichia coli (E. coli) vaccines containing 100 µg/ml of the recombinant mutant rETX^H106P/CTB-rETX^Y196E proteins mixed with different adjuvants. Apart from rETX^H106P/CTB-rETX^Y196E-IMS1313-vaccinated rabbits, the neutralizing antibody titers of rETX^H106P/CTB-rETX^Y196E-vaccinated rabbits were higher after the initial immunization than those administered the ETX toxoid or current commercial vaccines. rETX^H106P/CTB-rETX^Y196E mixed with ISA201 induced the highest neutralizing antibody titer of 120 after the first immunization, suggesting that 0.1 ml of pooled sera could neutralize 120× mouse LD₁₀₀ (100% lethal dose) of ETX. Following the second vaccination, rETX^H106P/CTB-rETX^Y196E mixed with ISA201 or GR208 produced the highest neutralizing titer of 800. Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD₁₀₀ of ETX challenge. These results show that these novel recombinant proteins can induce a strong immune response and represent potential targets for the development of a commercial vaccine against the C. perfringens epsilon toxin.     

Resistance of Ovine,Caprine and Bovine Endothelial Cells to Clostridium perfringens Type D Epsilon Toxin In Vitro

Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD⁵⁰/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD⁵⁰/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD⁵⁰/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro.     

Death of a neonatal lamb due to Clostridium perfringens type B in New Zealand

ABSTRACT

Case history and clinical findings: A flock of 20 sheep was kept within three paddocks on a single property. None of the animals in the flock had been vaccinated against any disease for at least three years. Abdominal bloating and haemorrhagic diarrhoea were observed in Lamb 1 at 24 hours-of-age. The lamb subsequently died within an hour of the onset of clinical signs. Lamb 2 was 3-days-old when observed to be recumbent with opisthotonus. The lamb was treated with dextrose, vitamins B1 and B12, and penicillin G, but died 4 hours later.

Pathological findings: Examination of Lamb 1 revealed markedly increased gas within the peritoneum and within dilated loops of intestine. The intestines were dark red and contained large quantities of haemorrhagic fluid. Histology of the intestines revealed peracute mucosal necrosis with minimal accompanying inflammation. The intestinal lumen contained cell debris, haemorrhage, and myriad large Gram-positive bacilli. The intestines of Lamb 2 did not appear bloated or reddened. However, multiple fibrin clots were visible within the pericardial sac. Histopathological examination revealed small foci of necrosis within the mucosa of the distal intestine. The necrotic foci were often associated with large numbers of large Gram-positive bacilli.

Immunohistochemsitry and molecular biology: Intestinal samples from Lamb 1 were processed for Clostridium perfringens immunohistochemistry, which revealed large numbers of intralesional, positively immunostained rods. Fragments corresponding to the expected sizes for genes encoding alpha, beta, and epsilon C. perfringens typing toxins were amplified by PCR from DNA extracted from formalin-fixed sections of intestine.

Diagnosis: Lamb dysentery due to C. perfringens type B.

Clinical relevance: C. perfringens bacteria have a worldwide distribution, but disease due to C. perfringens type B has only been diagnosed in a small number of countries and has never been reported in New Zealand or Australia. C. perfringens type B produce both beta toxin and epsilon toxins, therefore both haemorrhagic enteritis and systemic vascular damage can develop. As many animals are exposed to C. perfringens without developing disease, there must be additional unknown factors that resulted in disease in these particular sheep. Vaccines that specifically protect against C. perfringens type B are available and may be recommended for use in smaller non-commercial flocks, as in the present case.     

抗A型产气荚膜梭菌α毒素单链抗体基因的表达

将 2种抗 A型产气荚膜梭菌α毒素单链抗体 ( Sc Fv)基因 Sc Fv-2 E3和 Sc Fv-1 A8克隆至表达载体p UC1 1 9、p HOG2 1和 p ET2 0 b中 ,构建了重组质粒 p UC2 E3和 p UC1 A8、p HOG2 E3和 p HOG1 A8、p ET2 E3和 p ET1 A8后 ,分别转化至受体菌 JM1 0 5、JM1 0 9和 BL2 1 ( DE3 )中 ,得到重组菌株 JM1 0 5( p UC2 E3 )和JM1 0 5( p UC1 A8)、JM1 0 9( p HOG2 E3 )和 JM1 0 9( p HOG1 A8)及 BL2 1 ( DE3 ) ( p ET2 E3 )和 BL2 1 ( DE3 )( p ET1 A8)。 ELISA检测表明 ,经 IPTG诱导后所表达的目的蛋白存在于重组菌株 JM1 0 5( p UC2 E3 )和JM1 0 5( p UC1 A8)及 JM1 0 9( p HOG2 E3 )和 JM1 0 9( p HOG1 A8)的菌培养上清中 ,在重组菌株 BL 2 1 ( DE3 )( p ET2 E3 )和 BL2 1 ( DE3 ) ( p ET1 A8)的胞周质中检测到了 Sc Fv的存在。 SDS-PAGE和薄层扫描分析表明 ,重组菌株 BL2 1 ( DE3 ) ( p ET2 E3 )和 BL2 1 ( DE3 ) ( p ET1 A8)的蛋白表达产物分别占菌体可溶性蛋白的 0 .9%和 0 .7%,其大小约为 2 60 0 0 ,且表达的目的蛋白具有中和磷脂酶 C的活性。     

产气荚膜梭菌主要致死性毒素的研究进展

产气荚膜梭菌作为危害各国家畜养殖业的主要致病菌之一,受到国内外研究者的普遍关注.产气荚膜梭菌的主要致病因子是菌体产生的外毒素.近年来,国内外对其致病机理进行了深入研究,建立起简便、快速的检测方法,并且在防治手段方面取得了巨大突破.对产气荚膜梭菌的主要致死性毒素类型、致病机理、检测技术及其防治措施等方面的研究进展进行了阐述.     

羊源A型产气荚膜梭菌的分离鉴定与进化树分析

无菌采取内蒙古通辽市某羊场病死羊肠道内容物、肝脏和肺脏,进行细菌的分离培养。将从十二指肠内分离到的1株疑似致病菌株进行生化试验、小鼠致病性试验,再将其通过魏氏梭菌多重PCR试验、魏氏梭菌ELISA试验及16S rRNA PCR试验进行鉴定。将PCR产物进行测序并进行了16S rRNA基因的进化树分析。结果显示,经细菌生化试验、多重PCR试验、ELISA试验和16S rRNA试验均证实此分离株为A型产气荚膜梭菌;进化树分析显示该菌与序列号为HQ808749.1(美国)的A型产气荚膜梭菌遗传距离最近。结果表明,该羊病例所分离的致病菌为A型产气荚膜梭菌。