体外血脑屏障模型的构建及致脑膜炎粪肠球菌对其的影响

试验旨在探索致脑膜炎粪肠球菌对体外血脑屏障功能的影响，寻求稳定的构建体外血脑屏障损伤模型的方法。选用1周龄ICR小鼠进行原代脑微血管内皮细胞（BMEC）分离并通过BMEC特有的凝血因子Ⅷ（coagulation factor Ⅷ，Factor Ⅷ）进行荧光标记，鉴定分离的细胞并判定分离率。选用1~3日龄ICR小鼠分离星形胶质细胞并通过其特有的胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）对进行荧光标记，鉴定分离的细胞并判定分离率。将原代细胞传至3代，用Transwell二维小孔分别构建单层BMEC和BMEC与星形胶质细胞共培养的血脑屏障模型。选用致脑膜炎粪肠球菌、非致脑膜炎粪肠球菌和大肠杆菌DH5α感受态细胞分别与构建的体外血脑屏障模型共培养，评估致脑膜炎粪肠球菌对体外血脑屏障模型的穿透性。通过4 h渗透试验、荧光素钠通透性试验和血脑屏障损伤相关标志基因基质金属蛋白酶2（MMP-2）表达量的检测，评估致脑膜炎粪肠球菌对体外血脑屏障模型的影响。结果显示，试验成功分离到BMEC和星形胶质细胞，纯度分别达90%和95%以上。细菌穿透试验结果显示，所选的3株细菌只有致脑膜炎粪肠球菌可穿越体外血脑屏障模型。4 h渗透试验和荧光素钠通透性试验结果显示，共培养模型优于单层BMEC模型。与其他组相比，构建的体外血脑屏障与致脑膜炎粪肠球菌共培养组荧光素钠的穿透力更高，MMP-2基因的表达量明显上升。综上，BMEC与星形胶质细胞共培养的模型优于单层BMEC模型，致脑膜炎粪肠球菌可穿越体外血脑屏障模型，并介导体外血脑屏障损伤，本试验结果为揭示致脑膜炎粪肠球菌对体外血脑屏障损伤机制提供了理论依据。     

小鼠抗HCMV-gB多克隆抗体的制备及初步应用

为了快速制备抗HCMV-gB小鼠多克隆抗体,通过PCR扩增HCMV-gB(57aa～146aa)编码序列并克隆到原核表达载体pET21b(+)多克隆位点,用重组质粒转化大肠埃希菌Rosetta(DE3)并通过IPTG诱导表达,经镍胶亲和层析纯化和透析复性获得重组蛋白。以CpG-ODN和Al(OH)3复合佐剂作为重组蛋白的免疫佐剂,通过0周和3周2次肌肉注射免疫Balb/c小鼠,并在第5周采血分离免疫血清。用ELISA检测免疫血清效价,并通过Western blot检测免疫血清对哺乳动物细胞表达的HCMV-gB1和gB2的反应性。结果显示,本研究制备的免疫血清ELISA滴度达到1∶51 200～1∶102 400,进行Western blot能够与哺乳动物细胞表达的HCMV-gB1和gB2发生特异性反应。此试验获得了能够用于后续研究工作的抗HCMV-gB小鼠多克隆抗体,本方法具有制备速度快、抗体滴度高和抗原用量少等优点。     

SS2溶菌酶释放蛋白(MRP)B细胞表位预测、原核表达及免疫学特性

运用ANTHEPROT 5.0综合分析二级结构、亲水性、表面可及性与抗原性指数,预测MRP的B细胞优势袁位簇;PCR扩增出表达预测的抗原肽段的基因(mrp-1),构建重组原核表达载体pET32a-mrp-1,IPTG诱导表达,以渗透性休克法纯化重组蛋白;使用重组蛋白免疫血清和灭活猪链球菌2型SS2免疫血清Western blotting检测重组蛋白的免疫原性及其与SS2免疫血清的反应性;纯化的重组蛋白免疫雌性SPF昆明小鼠,SS2攻毒受试动物评估重组蛋白对小鼠的免疫保护力.Western blotting显示预测的950～1 210 aa肽段与PET32a重组表达的蛋白(MRP)具有良好的免疫原性和反应性;攻毒试验结果表明重组蛋白对小鼠的免疫保护率为菌体免疫所提供保护率的66.7％.研究为利用该细胞表位簇作为高效免疫制剂奠定了基础.     

产肠毒素大肠杆菌STa突变体、LTB和STb融合蛋白在乳酸菌中组成型分泌表达及其免疫原性分析

为获得产肠毒素大肠杆菌(ETEC)抗原STa突变体(mSTa)、LTb和STb融合蛋白在乳酸菌表达系统中组成型分泌表达,本研究将mSTa、LTb、STb三价抗原融合基因,克隆于p23启动子-USP45分泌信号肽之后,插入到乳酸乳球菌表达载体pTX8048中,构建了重组表达载体pTX-sls,将其电转化到宿主菌乳酸乳球菌L.lactisNZ9000中进行表达,应用westernblot、ELISA方法鉴定蛋白表达情况。将重组乳酸乳球菌pTX-sls/NZ9000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;采用MTT法检测免疫小鼠脾淋巴细胞增殖情况,流式细胞术检测Th细胞免疫类型。结果显示,目的蛋白mSTa-LTB-STb以分泌形式组成型表达,可被阳性血清所识别。在免疫小鼠血清和粪便中均可检测到特异性IgG、sIgA,血清抗体具有一定的中和毒素作用。结果表明,该重组乳酸乳球菌具有作为口服疫苗的潜在应用价值,本研究为研制ETEC乳酸菌活载体口服疫苗奠定了基础。     

微小隐孢子虫黏附相关蛋白基因CP21的原核表达及鉴定

本研究旨在克隆微小隐孢子虫黏附相关蛋白基因CP21开放阅读框,构建其原核表达质粒,并对获得的融合蛋白进行鉴定。从含CP21基因的噬菌体中提取模板DNA,PCR扩增基因片段,构建pET-28a-CP21原核表达质粒,转化至埃希氏大肠杆菌BL21(DE3)中,IPTG诱导表达,表达产物经SDS-PAGE和Western blotting分析鉴定。用原核表达蛋白免疫小鼠,制备抗重组蛋白多抗,对蛋白进行定位。结果表明成功扩增出CP21基因,并构建原核表达质粒,转化至大肠杆菌中表达出相对分子质量为25 ku的融合蛋白,Western blotting证明表达的CP21融合蛋白能与抗C.parvum多克隆抗体产生特异性反应。免疫荧光结果表明:CP21基因在子孢子和卵囊期均表达,且表达产物位于子孢子表膜和卵囊壁表面。CP21是一种与侵入机制有关的黏附相关蛋白。研究结果为微小隐孢子虫的免疫学诊断及疫苗研制奠定了基础。     

短小乳杆菌重组S-层蛋白质的纯化及其体外黏附性的初步鉴定

以本实验室已构建的pMD19T-slp为模板,应用PCR方法亚克隆slp基因,将其定点插入到原核表达载体pGEX-4T-3中,成功构建原核表达载体pGEX-slp,经IPTG诱导获得重组融合蛋白GST-SLP并用LiCl的方法进行纯化。应用SDS-PAGE和Western blot方法对表达产物进行鉴定,应用ELISA方法鉴定重组融合蛋白的体外黏附特性。在大肠埃希菌裂解样品中,SDS-PAGE和Western blot结果均证明分子质量大小约为71ku的重组融合蛋白GST-SLP的存在;ELISA结果提示,该重组融合蛋白在体外能够有效地黏附在乳酸乳球菌NZ9000表面。本试验为S-层蛋白质生物学特性的进一步研究奠定了基础。     

猪流行性腹泻病毒COE核酸疫苗的构建及免疫原性

用疑似患猪流行性腹泻病猪肠病料,扩增部分保护性抗原基因（COE基因）,通过T4连接酶将COE基因与真核表达载体plRES2-EGFP进行连接,提取纯化重组质粒。通过脂质体转染vero细胞,用SDS-PAGE和westernblot鉴定COE蛋白的表达。用重组质粒对BALB／c小鼠进行免疫,观察免疫效果。结果显示成功构建了pIRES2-EGFP—COE真核表达载体;目的蛋白在vero细胞中得到表达;重组质粒免疫小鼠能够产生相应抗体。结果表明,COE核酸疫苗可在小鼠体内诱导相应的抗体产生,这为进一步研究猪腹泻病毒的核酸疫苗奠定了基础。     

猪致病性大肠埃希菌外膜蛋白亚单位疫苗免疫效果观察

用超声波破碎并采用N-十二烷基肌氨酸钠进行猪致病性大肠埃希菌外膜蛋白提取,提取物经SDS-PAGE电泳检测后制备成疫苗,以肌肉注射方式免疫SPF小鼠,于免疫前及免疫后每隔1周分别收集血清.以间接ELISA的方法检测血清中IgG抗体水平,同时用不同血清型大肠埃希菌菌株与免疫血清做玻板凝集试验和攻毒保护试验.结果表明,在免疫小鼠后的第28天左右机体内的抗体水平达到最高值,免疫血清能够与不同菌株发生凝集反应且不同菌株对小鼠的攻毒都具有免疫保护力,说明外膜蛋白不仅具有一定的免疫原性而且对不同菌株具有交叉免疫保护力,因此外膜蛋白亚单位疫苗作为一种新型疫苗可望适用于防控猪大肠杆菌病.     

鸡传染性支气管炎病毒QX基因型S1蛋白重组乳酸菌的构建及小鼠免疫应答

为探究鸡传染性支气管炎病毒(IBV)S1蛋白重组乳酸菌对小鼠免疫应答情况,本研究将IBV CH/LN/2019毒株的S1基因进行密码子优化,构建重组质粒pNZ8149-S1,电转至乳酸乳球菌NZ3900中,应用Western blot及间接免疫荧光试验评价重组乳酸乳球菌表达水平,免疫SPF小鼠,通过间接ELISA方法分析小鼠特异性抗体、CD4⁺、CD8⁺和细胞因子(IL-4和IFN-γ)分泌情况;结果显示,成功构建重组乳酸乳球菌pNZ8149-S1/NZ3900并表达目的蛋白;小鼠免疫试验结果显示,免疫组小鼠IgG抗体水平均高于pNZ8149/NZ3900、NZ3900和PBS组(P<0.05);特异性sIgA抗体水平显著高于对照组(P<0.01);此外,重组乳酸乳球菌能诱导小鼠产生较高水平的IL-4和IFN-γ(P<0.05)。结果表明,重组乳酸乳球菌pNZ8149-S1/NZ3900可以有效刺激小鼠机体产生体液免疫和细胞免疫,为IBV新型口服疫苗的研制提供了理论参考。     

奶牛隐性乳房炎奶样中埃希氏大肠杆菌共同性抗原初探

用LMT的方法对新疆8个奶牛场采集的1020份奶样进行隐性乳房炎的检测,并对阳性样品进行了大肠杆菌的分离与鉴定。从不同牛场分离菌株中挑选8株用家兔制备免疫血清,用试管凝集对从乳房炎奶样分离出的其它21株大肠杆菌进行共同性抗原的筛选。结果显示,免疫血清效价达1∶2560～1∶5120,85.7%的待检菌株与其中3种血清凝集反应呈阳性。对制备血清的8株菌进行SDS-PAGE电泳分析,菌体蛋白图谱显示在20~84 ku处存在大量相同或相似的蛋白条带;用制备的免疫血清进行免疫印迹分析,与8株菌的菌体蛋白在70 ku处呈现明显的抗原抗体反应。说明这些菌株存在共同性抗原。     

A RADIOGRAPHIC STUDY OF THE DISTAL INTERPHALANGEAL JOINT AND NAVICULAR BURSA OF THE HORSE

Radiographic contrast studies were used in 50 forelimbs from 13 live horses and 12 fresh adult cadavers to determine the frequency of communication between the navicular bursa and the distal interphalangeal joint. Injections of contrast medium were made into the dorsal aspect of the distal interphalangeal joint of one limb and into the navicular bursa of the other forelimb of each horse. In 25 limbs in which contrast medium was injected into the distal interphalangeal joint, no communication was demonstrated between the joint and the navicular bursa. In 20 of the 25 limbs in which injection was made into the navicular bursa, no communication between joint and bursa was seen. In five horses, contrast medium was visible in both the distal interphalangeal joint and the navicular bursa. However, in four of five horses the communication was clearly iatrogenic. In both limbs of one horse, contrast medium was seen to enter the digital flexor tendon sheath after injection into the navicular bursa.
There is probably no naturally occurring communication between the navicular bursa and distal interphalangeal joint in the horse.     

精原干细胞介导法制备转基因羊的研究进展

精原干细胞介导法制备转基因动物是用试验导入的方法将外源基因移入动物细胞并整合到其基因组中,伴随着精原干细胞分化成精子,通过受精最终使外源基因得以表达。近年来,随着对精原干细胞研究的不断深入,人们发现其在建立转基因动物方面具有巨大的应用潜力和优势。作者从精原干细胞的生物学特性及携带外源基因的原理等方面阐述了其应用于转基因动物制备的理论机制,同时介绍了精原干细胞介导法在制备转基因动物方面,特别是转基因羊生产中的应用现状。     

法氏囊活性肽在大肠杆菌中的克隆与表达

法氏囊活性肽（Bursin,BS)作为一种免疫增强剂，临床应用广泛，为了大量制备BS，按大肠杆菌惯用密码子合成BS基因。并用色氨酸将5个BS基因串联，克隆于质粒pU57的SmaI位点，经测序证明序列正确后，以PCR扩增，克隆于表达载体pBV220的EcoRI,HinDⅢ位点，温敏诱导表达，其表达水平占全菌总蛋白的13.4%。     

Local immune response in the chicken Harderian gland to antigen given by different ocular routes.

The effectiveness of three ocular routes of antigen administration to produce a local immune response in the Harderian gland was studied. The routes were by eyedrop, injection into the ocular conjunctiva and injection into the nictitating membrane. The antigen was observed in the cytoplasm of macrophages located within the lymphoid tissue only after the injection into the nictitating membrane. The numbers of germinal centres and plaque forming cells found in the gland after injection into the nictitating membrane was higher than the numbers observed following the other two ocular applications. These findings indicate that the injection of the antigen into the nictitating membrane is the most effective ocular route for producing a local immune response in the Harderian gland.     

水貂肠炎病毒VP2基因在昆虫杆状病毒表达系统中的表达

利用昆虫杆状病毒表达系统表达水貂肠炎病毒(mink enteritis virus,MEV) VP2 基因,表达的蛋白能够形成病毒样粒子,具有反应原性,为研究MEV新型疫苗奠定基础。采用PCR方法扩增MEV VP2 基因,将PCR产物连接到pMD18-T载体,目的基因定向克隆到pFast-BacⅠ载体中,构建重组转座载体后转化DH10 Bac感受态细胞,获得重组Bacmid 质粒后转染Sf9昆虫细胞,传毒3代,对表达蛋白进行 Western blotting鉴定。结果成功克隆MEV VP2基因。Western blotting结果证实,表达蛋白能够被MEV单克隆抗体识别。在 Bac-To-Bac 杆状病毒表达系统中成功的表达了MEV VP2蛋白,目的蛋白具有较好的反应原性。     

Characteristic transfer of colostrum-derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs

The characteristic transfer of colostral components into cerebrospinal fluid (CSF) via serum after natural suckling has been studied by sodium dodecyl sulphate (SDS) electrophoresis, two-dimensional electrophoresis, immunoblot and enzyme linked immunosorbent assay methods in non-suckling pigs. Total protein concentrations in the serum increased immediately after first suckling, reached a peak value at 12 h, corresponding to a 2.3-fold compared with pre-suckling level. The protein concentration in CSF also increased and reached peak value at 6 h corresponding to 1.6-fold compared with presuckling level. IgG in serum not detected before suckling, increased steeply after suckling, IgG, IgM and IgA transported into the serum were observed in completely intact form by immunoblot method. The IgG transported into serum was quickly transferred into CSF after natural suckling in contrast to the case of bovine IgG. Serum concentration of transferrin was maintained at high level before suckling and was not changed by suckling. Transferrin also detected in CSF was not changed by suckling. Bovine lactoferrin (Lf) administered into the intestinal lumen was transported into serum (0.01%) and also detected in CSF after 6 h as undegraded form (3.1%). Thus, homologous IgG and bovine Lf are transported into CSF, suggesting that the transport of macromolecules into CSF is selective in neonatal pigs.     

黑曲霉糖化酶基因在毕赤酵母X33中的高效表达

为高效表达黑曲霉糖化酶基因,根据GenBank中糖化酶的氨基酸序列(登录号:AY652617),选用毕赤酵母(Pichia pastoris)密码子偏嗜性,全基因合成新的cDNA序列。改造后的基因克隆到pGAPZαA质粒中,获得重组分泌型酵母表达质粒pGAPZαA-EC,经限制性内切酶Bln Ⅰ酶切线性化后,电击转化入毕赤酵母细胞X33内。经高浓度博莱霉素抗性筛选,得到高拷贝转化子,PCR检测结果显示,糖化酶基因与毕赤酵母染色体稳定结合。糖化酶蛋白获得分泌表达,SDS-PAGE分析其分子质量约为80 ku,其表达量约为180 mg/L。表达产物经Starch-PAGE活性染色,显示其具有酶学活性。     

Glycerolipid biosynthesis in porcine adipose tissue in vitro. I. Assay conditions for homogenates

Previous studies on glycerolipid biosynthesis in swine adipose tissue in vitro resulted in synthesis of primarily phospholipid, whereas triacylglycerol represents the vast majority of adipose tissue lipids. The objectives of this research were to maximize synthesis of triacylglycerol in vitro using the 700 x g infranatant fraction of a swine adipose tissue homogenate as the enzyme source. The capacity for total lipid synthesis was increased by greater than 50%, and the proportion of lipids synthesized as triacylglycerols was increased by increasing the length of incubation time from 20 to 60 min and the concentration of enzyme in the incubation from that obtained from 33 to that obtained from 120 mg adipose tissue. It is recommended that glycerolipid biosynthesis be assessed using two assays. An assay of up to 10 min was linear with incubation time and measured the initial incorporation of glycerol-3-phosphate into the pathway (GPAT); this incorporation was mostly into phospholipids. An assay of about 60 min was not linear with incubation time, but incorporation into total lipids (LSC) was predominantly into the triacylglycerol fraction. Although the LSC assay was not linear with time, it represents steady-state conditions that more closely typify conditions in situ. Oleate at .6 mM was inhibitory with enzyme extracted from 33 or 75 mg adipose tissue, whereas palmitate was not. Palmitoyl-CoA was not a suitable substrate because it produced low LSC and little triacylglycerol. Fluoride increased LSC but inhibited conversion of phospholipids into triacylglycerols, so its presence is not recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of Homoarginine into the Gut of Chickens

Angkanaporn, K., Ravindran, V., Mollah, Y. and Bryden, W.L., 1997. Secretion of homoarginine into the gut of chickens. Veterinary Research Communications, 21(3), 161-167A technique, based on the homoarginine present in guanidinated proteins, has been used to distinguish between endogenous secretions and exogenous dietary amino acids in the ileal digesta of monogastric animals. This technique assumes that the ingested homoarginine is not recycled into the small intestine after absorption, but this assumption is yet to be experimentally validated in chickens. The secretion of homoarginine into the gut of broilers that were intravenously infused with 20 and 40 mmol/L homoarginine solutions was assessed. The plasma concentrations of homoarginine increased with increasing concentrations of homoarginine infused. However, only negligible levels of homoarginine (7.0 to 45.2 µg/g dry matter) were found in the digesta. Less than 0.01% of the intravenously infused homoarginine was recovered in the intestinal digesta, indicating that the secretion of homoarginine into the gut of chickens was insignificant.     

猪源多杀性巴氏杆菌ompH基因的克隆、表达

利用已分离的菌株030224HB，根据NCBI上的序列(U52208)设计了一对引物，用PCR方法扩增了猪源多杀性巴氏杆菌的外膜蛋白基因(ompH)，扩增的片段大小为1114bp(ORF为960bp)，并克隆到载体pMD18-T(T-Vector)，测序表明该基因相当保守。用pET-28b构建了原核表达载体pET28b-ompH，转化BL21并诱导表达，SDS-PAGE结果显示表达蛋白约为35ku，与报道大小相近。Western-blot结果表明表达的蛋白质具有生物学活性，然后用所表达的蛋白做了ELISA检测方法的初步探讨。