鼠李糖乳杆菌菌毛SpaA亚基的表达、抗体制备及其种属特异性研究

【目的】制备鼠李糖乳杆菌菌毛亚基Spa A多克隆抗体,研究其种属特异性。【方法】应用PCR方法从鼠李糖乳杆菌GG的基因组扩增出spa A,并连接到质粒p ET-28α(+)中。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达和镍柱纯化制备重组SpaA。通过免疫BALB/c小鼠获得多克隆抗体,利用全菌ELISA、Western和Dot-blot分析了SpaA在18株乳酸菌(12个种)中的分布特征。【结果】表达的重组SpaA分子量为36 k D,与预期大小一致;获得的Spa A抗体效价为1:12 800。Western结果显示抗体与天然Spa A具有良好的反应性。在测定的18株乳酸菌中,鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌3个种属菌株的spa A基因PCR和RT-PCR检测均为阳性。但全菌ELISA和Dot-blot结果显示,只有3株鼠李糖乳杆菌的全菌细胞与SpaA抗体呈特异性反应,而其它种属的菌株没有明显的交叉反应。【结论】尽管spa A基因在鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌中具有高度同源性,但SpaA蛋白只特异性地呈现在鼠李糖乳杆菌细胞表面。本研究中获得的Spa A抗体,为高黏附性鼠李糖乳杆菌的免疫磁珠分离及菌毛功能研究提供了工具。     

表达ETEC F41重组干酪乳杆菌口服及滴鼻免疫效果的比较

摘要：【目的】比较小鼠滴鼻与口服接种ETEC F41重组干酪乳杆菌后,机体所产生的抗ETEC F41的黏膜免疫和系统免疫及免疫保护率的差异,为确定ETEC乳酸菌疫苗免疫程序奠定基础。【方法】将构建的重组质粒pLA-F41电转化入干酪乳杆菌,获得阳性重组菌。重组菌在MRS 培养基中进行表达,经Western blot检测目的蛋白的表达,间接免疫荧光及流式细胞术检测外源蛋白展示到菌体表面。SPF级BALB/c小鼠随机分成4组,每组40只,将重组菌以滴鼻和口服途径分别接种2组小鼠,对照组分别接种同剂量的空质粒菌     

猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白在干酪乳杆菌中的共表达及免疫小鼠特异性抗体的测定

将分别编码猪瘟病毒T细胞表位E290多肽和猪细小病毒主要保护性抗原VP2蛋白的重组基因插入干酪乳杆菌分泌型表达载体pPG中,构建了重组表达载体pPG-VP2-E290,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白的乳酸菌共表达系统,经2%乳糖在MRS培养基中的诱导表达,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约70kDa蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot分析结果表明所表达的蛋白具有与天然病毒蛋白一样的抗原特异性。以诱导表达上清液作为抗原进行的间接ELISA实验也表明,重组的目的蛋白获得了分泌表达。将该重组干酪乳杆菌经口服接种途径免疫BALB/c小鼠,收集粪便样品检测小鼠产生抗PPV的特异性sIgA抗体,采集血液样本检测血清中抗PPV及抗E290的特异性IgG。结果表明分泌型的重组菌pPG-VP2-E290/L.casei 393免疫小鼠能够产生明显的抗体水平,为重组猪瘟与猪细小病毒乳酸菌口服活菌疫苗的研制奠定了重要的物质基础。     

人胰岛素基因在乳酸菌中的表达及其对非肥胖糖尿病(NOD)小鼠的作用

为实现人胰岛素基因在乳酸菌中的表达及探索其用作口服疫苗治疗Ⅰ型糖尿病(T1DM)的可行性,首先将人胰岛素基因密码子替换为乳酸菌偏爱密码子,同时在A,B链序列间加入连接短肽序列,经引物退火拼接合成人胰岛素基因。克隆至乳酸菌表达载体中后,利用电击转化法实现了带信号肽SPUsp45的人胰岛素基因在乳酸乳球菌(Lactococcus lactis)MG1363和干酪乳杆菌(Lactobacillus casei)ATCC27092中的表达。Western blot检测显示重组胰岛素位于细胞壁上,当菌体生长到OD600为0.4时达到最大表达量。用含有表达重组人胰岛素的Lactobacillus caseiATCC27092/pSW501菌体饲喂非肥胖糖尿病(NOD)小鼠,发现可刺激小鼠产生特异性抗体,同时使与免疫耐受相关的细胞因子IL-4水平明显升高(38.583±2.083pg/mL,P<0.05),提示其对NOD小鼠产生免疫耐受有一定的作用,为研制乳酸菌口服疫苗防治T1DM的可行性进行了有益的探索。     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及其小鼠口服免疫力

[目的]构建产气荚膜梭菌(Clostridium perfringens,C.perfringents)α毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法.[方法]将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌(Lactobacillus casei L.casei),获得阳性重组菌pPG1-α/L.case/393乳酸乳杆菌表面表达系统和pPG2-α/L.casei 393乳酸乳杆菌分泌表达系统.重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达.将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性slgA抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性lgG抗体水平.并对免疫小鼠进行α毒素的腹腔攻毒试验及对获得的抗血清进行α毒素中和试验测定.[结果]重组干酪乳杆菌pPG1-α/L.casei 393及pPG2-/L.casei 393免疫小鼠能够产生明显的抗α毒素的slgA和IgG抗体水平,其对α毒素中和试验结果为完全保护.腹腔攻毒实验结果为能抵抗3倍最小致死剂量(minimum lethal dose,MLD100)的α毒素攻击.[结论]表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力.     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及小鼠口服免疫保护效力的测定

摘要: 【目的】构建产气荚膜梭菌（Clostridium perfringens, C．perfringens）α 毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法。【方法】将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌（Lactobacillus casei L.casei）,获得阳性重组菌pPG1-α/ L.casei 393 乳酸乳杆菌表面表达系统和pPG2-α/ L.casei393乳酸乳杆菌分泌表达系统。重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达。将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性sIgA 抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性IgG抗体水平。并对免疫小鼠进行α毒素的腹腔攻毒实验及对获得的抗血清进行α毒素中和试验测定。【结果】重组干酪乳杆菌pPG1-α/ L.casei 393及pPG2-/ L.casei 393免疫小鼠能够产生明显的抗α毒素的sIgA 和IgG 抗体水平,其对α毒素中和试验结果为完全保护。腹腔攻毒实验结果为能抵抗3倍最小致死剂量的α毒素攻击。【结论】表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力。     

猪源A组轮状病毒重组质粒pW425et-Vp7在乳酸菌中的表达及免疫原性分析

[目的]在乳酸菌中表达猪源A组轮状病毒重组质粒pW425et-Vp7,并检测其抗原性,为进一步制备猪轮状病毒乳酸菌疫苗奠定基础.[方法]摸索电转化条件,将已构建的重组质粒pW425et-Vp7转化至thyA基因缺陷型的嗜酸乳杆菌中.筛选阳性克隆,采用SDS-PAGE和Westernblot检测其表达情况.并将BALB/c小鼠口服免疫该重组菌,间接ELISA检测其免疫原性.另外,给免疫后小鼠攻以纯化的猪轮状病毒(蛋白含量15 mg/mL),初步检测该重组菌的免疫保护作用.[结果]Vp7基因在乳酸菌中获得表达,大小约40.77 kDa,且该蛋白可与猪轮状病毒抗体反应,具有反应原性.ELISA检测小鼠肠黏膜样品结果显示免疫后小鼠能产生明显的抗轮状病毒SIgA抗体,表明该重组菌具有较好的免疫原性.且攻毒后,免疫组仅23%的小鼠发生腹泻,初步证明该重组菌对猪轮状病毒的攻击具有较好的保护作用.[结论]在乳酸菌中成功地表达了猪轮状病毒Vp7基因,制备了猪轮状病毒Vp7重组乳酸菌,该重组菌具有较好的抗原性,初步证明其作为疫苗的可行性.     

重组GFP干酪乳杆菌的构建及其在小鼠肠道内的定植分布

【目的】研究重组干酪乳杆菌(Lactobacillus casei)在小鼠肠道内定植能力及分布规律。【方法】利用绿色荧光蛋白(GFP)基因作为报告基因,构建pgsA基因与GFP的融合基因载体pLA-GFP,电转化到乳酸杆菌中,得到阳性重组菌。将重组菌以每只109mL-1的量,口服接种SPF级BALB/c小鼠,分别于口服后的1.5h、3h、12h、1d、3d、5d、6d、7d之后取其十二指肠、空肠、回肠、盲肠的肠道冲洗液,通过平板菌落计数法检测肠道内的重组干酪乳杆菌。【结果】Western blot结果显示约69kDa的融合蛋白在乳酸菌中得到了正确的表达;重组菌在蓝紫光激发下,发出绿色荧光。小鼠口服重组菌后能在肠道黏膜的不同部位以一定的比例存活并附着在肠黏膜表面,口服6d后达到定植高峰期,7d后在十二指肠、空肠、回肠和盲肠定植率分别占第1天的16.49%、25.08%、47.71%、41.03%。【结论】GFP在干酪乳杆菌中得到了稳定的表达,且在小鼠肠道内具有良好的定植能力,定植规律回肠盲肠空肠十二指肠,这为研究乳酸杆菌作为口服疫苗抗原递送载体及其对小鼠肠道免疫机理提供试验基础。     

表面表达猪流行性腹泻病毒核蛋白蛋白的重组干酪乳杆菌诱导产生的免疫应答

为探讨表达猪流行性腹泻病毒(PEDV)核蛋白(N)基因的重组干酪乳杆菌口服免疫小鼠后诱导特异性免疫应答,本研究制备表达流行性腹泻病毒核蛋白的重组干酪乳杆菌,应用Western blotting、间接免疫荧光和全细胞ELISA鉴定目的蛋白的表达。然后用该重组干酪乳杆菌口服免疫BALB/c小鼠,分别测定了免疫后不同时间血清中特异性IgG、粪便中特异性的sIgA水平以及血清的中和活性;并测定免疫小鼠脾淋巴细胞增殖情况和细胞因子水平。结果显示,目的蛋白表达在细胞表面,可被阳性血清所识别。免疫小鼠后,可分别在血清中和粪便中检测到较高水平特异性IgG、sIgA(P<0.01),但血清并没有中和活性;淋巴细胞增殖试验和细胞因子测定结果显示,免疫组可产生明显的细胞免疫应答。结果表明,该重组干酪乳杆菌表达系统可诱导小鼠产生黏膜免疫应答和系统免疫应答,具有作为口服疫苗潜在的应用价值。     

p LA-PEDV-S1质粒拷贝数与发酵重组乳酸菌S1表达量之间的相关性研究

探究质粒拷贝数以及目标蛋白S1表达量与发酵时间的关系,从而确定放大生产p LA-PEDV-S1/Lactobacillus casei的最佳发酵时间。通过发酵重组干酪乳杆菌,绘制重组干酪乳杆菌的生长曲线,确定其生长的最佳时期。将p LA-PEDV-S1/L. casei分别接种至添加抗生素和不添加抗生素的MRS培养基中传代培养,进行稳定性实验。使用荧光定量PCR方法检测重组干酪乳杆菌中质粒的拷贝数,使用流式细胞术检测乳酸菌表达的目标蛋白。重组菌在7 h达到生长顶点,传至120代时外源质粒并无丢失情况出现。质粒拷贝数在9 h达到峰值29. 34,表达目标蛋白的重组菌在7 h达到峰值97. 98%。结果显示在细菌生长的对数生长期末期,质粒拷贝数最高且S1表达量最多;在平台期随着发酵时间的增加,质粒拷贝数逐渐降低,S1表达量也相应减少。发酵的最佳时间为7～10 h,质粒拷贝数与S1表达量之间存在着正相关性。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).