香石竹斑驳病毒外壳蛋白基因的原核表达及抗血清制备

 香石竹斑驳病毒（Carnation mottle virus,CarMV）是侵染香石竹的主要病毒。本研究从已鉴定的CarMV毒源植物中提取总RNA, 克隆外壳蛋白（cp）基因后构建pET30a CP重组质粒,并转入BL21（DE3）pLysS进行原核表达。通过对诱导温度、初菌浓度、IPTG的浓度等表达条件进行优化,目的蛋白在37℃, IPTG 终浓度为10mmol/L的条件下,诱导表达6h后获得最高表达量。采用割胶的方法纯化cp蛋白,纯化后的目的蛋白免疫小鼠获得了特异性抗血清。经Western blot检测,结果表明所制备的抗血清与诱导表达的重组蛋白及接种CarMV的香石竹样品均发生特异性结合。利用所制备抗血清对昆明地区的48个香石竹样品进行抽检,结果表明香石竹样品的带毒率为79.2%。本研究为大规模抗血清生产、检测应用和深入研究该病毒cp基因与寄主的互作奠定基础。     

瓜类褪绿黄化病毒p22 基因在大肠杆菌中的表达及抗血清的制备

 以被瓜类褪绿黄化病毒（Cucurbit chlorotic yellows virus,CCYV）侵染的甜瓜叶片为供试材 料,采用RT-PCR 方法克隆其P22 蛋白基因,并将其连接到原核表达载体pGex-4T-3 上,PCR 验证及克隆 测序确定开放阅读框的正确性。将重组载体pGexp22 转化大肠杆菌BL21 菌株,诱导表达,SDS-PAGE 分 析表明,经IPTG 诱导,p22 基因在大肠杆菌BL21 中得到了高效表达。以表达的蛋白作为抗原,免疫家 兔,制备了CCYV P22 的特异性抗血清。ACP-ELISA 检测结果表明,血清效价高达1.28 × 10⁵。Western blot 检测甜瓜叶片,结果表明抗血清能够特异性地检测CCYV 侵染的甜瓜叶片中的CCYV P22 蛋白。     

免疫外科法分离山羊囊胚内细胞团的研究

分别用关中奶山羊脾脏细胞和胎儿成纤维细胞,免疫新西兰白兔各2只,共4只。通过3次静脉注射,每次注射4×108个细胞,间隔10 d,最后一次注射10 d后心脏采血制备抗血清,结果获得的4份抗血清均具有细胞毒性,由脾脏细胞免疫获得的抗血清溶解细胞的效价稍高。超排关中奶山羊24只和波尔山羊3只,6~9 d采胚共获得胚胎240枚,其中囊胚106枚,结果显示配种后7~9 d胚胎的发育阶段适宜于分离内细胞团(ICM)。细胞毒性试验表明,抗血清对关中奶山羊红细胞、囊胚和波尔山羊囊胚均具有细胞毒性作用。通过比较抗血清50%溶解山羊红细胞效价与溶解囊胚滋养层细胞效价的差异,发现后者的适宜效价是抗血清50%溶解山羊红细胞效价的22~23倍,表明采用易于获得的山羊红细胞作预试验可以为免疫外科分离山羊ICM提供抗血清稀释比例参考。免疫外科法分离ICM采用两步法进行,获得最佳参数如下:抗血清稀释比例为1∶4~1∶15,补体稀释比例为1∶20,先在抗血清中作用30 min,再在补体中作用20~30 min,吹打除去溶解的滋养层细胞后即可获得ICM。分离得到的ICM形态特征与胚胎质量密切相关。山羊ICM接种于丝裂霉素处理过的小鼠胎儿成纤维细胞饲养层后,2 d内贴壁。4~7 d后,ICM增殖为胚胎干细胞样集落,其周围出现大量空泡样细胞。     

大肠埃希氏菌F18菌毛结构蛋白与黏附特性的关系

利用已表达的F18ab和F18ac菌毛各结构亚单位的融合蛋白（GST-FedA／ab、GST-Fe-dA／ac、GST-FedE、（；STFed-F／ab、GST-FedF／ac）分组肌肉注射健康家兔，制备抗FedA／ab、Fe-dA／ac、FedE、FedF／ab、FedF／ac多价血清。结果表明，所制备的5种抗Fed多价血清均能凝集F18ab^＋。大肠埃希氏菌107／86株和F18ac^＋大肠埃希氏菌8813株。利用抗大肠埃希氏菌F18菌毛主要结构亚单位FedA特异抗体和次要结构亚单位FedE、FedF特异抗体，研究了F18菌毛与小肠上皮细胞的黏附特性。结果发现，FedA抗体和FedE抗体单独和合并均不能抑制F18^＋菌与小肠上皮细胞的黏附，而单用抗FedF抗体即能明显抑制F18^＋大肠埃希氏菌与小肠上皮细胞的黏附，表明FedA和FedE与F18菌毛的黏附不具相关性，而FedF才是F18菌毛的黏附性结构亚单位。     

禽腺联病毒Rep78和VP3蛋白的原核表达及抗血清制备

分别将禽腺联病毒(Avian adeno-associated virus,AAAV)的Rep78基因和VP3基因克隆入pET-47b原核表达载体并转化BL21(DE3)大肠杆菌,在IPTG的诱导下2种目的蛋白均成功得到了表达。SDS-PAGE显示,Rep78蛋白的相对分子质量约为85 000,而VP3蛋白相对分子质量约为60 000。Western-blot分析显示,表达产物均能与抗AAAV的阳性血清反应。将目的蛋白切胶免疫BALB/c小鼠分别制备了针对2种蛋白的多克隆血清。间接免疫荧光试验显示制备的抗血清能够与AAAV抗原特异反应。不与鸡胚致死孤儿病毒(CELO)抗原反应。结果表明,制备的抗Rep78和VP3蛋白的血清可以用于检测重组AAAV载体制备过程中Rep和Cap基因的表达水平。     

Cross-reactions of Corynebacterium sepedonicum antisera with soil bacteria associated with potato tubers

The occurrence of low numbers of fluorescing coryneform bacteria located by immunofluorescence microscopy (IF) in heel-end extracts of healthy potatoes was demonstrated to be a normal phenomenon. The number of bacteria found was highly dependent on the antiserum dilution. Bacteria isolated from other healthy potato material also cross-reacted, mostly at low antiserum dilutions compared with homologous isolates or bacteria of the same species, which reacted at a dilution of 11280. Only relatively small differences were found between the antisera tested. Failure to recognize the occurrence of cross-reacting unknown soil bacteria, indicated by the data presented in this paper, can only increase the dangers of incorrect interpretation of IF results.Samenvatting Het optreden van lage aantallen fluorescerende, coryneforme bacteriën gevonden met behulp van immuno fluorescentiemicroscopie in extracten van naveleinden van genzonde aardappelen, bleek een normaal verschijnsel te zijn. Het aantal gevonden bacteriën was sterk afhankelijk van de verdunning van het antiserum. Bacteriën die geïsoleerd werden van ander gezond aardappelmateriaal vertoonden ook kruisreacties, meestal bij lage verdunningen van het antiserum in vergelijking met homologe isolaten of bacteriën van dezelfde soort, die beide nog reageerden bij een verdunning van 11280. Tussen de getoetste antisera bleken slechts geringe onderlinge verschillen te bestaan. De gegevens in dit artikel onderstrepen het gevaar van een onjuiste interpretatie van IF-resultaten, wanneer het voorkomen van kruisreagerende, onbekende bodembacteriën over het hoofd wordt gezien.     

昆明地区香石竹斑驳病毒的鉴定、提纯及抗血清制备

在昆明地区栽培的香石竹上发现了香石竹斑驳病毒,人工接种可局部侵染苋色藜、墙生藜、千日红及番杏,可系统侵染昆诺阿藜和美国石竹,分别造成褪绿斑、斑驳或坏死斑。经二轮ＰＥＧ（分子量６０００）沉淀,病毒提纯量约２２０ｍｇ／ｋｇ鲜组织。电镜观察病毒颗粒直径为２８ｎｍ,等轴对称。提纯物紫外扫描呈典型病毒核蛋白吸收峰,Ａ２６０／Ａ２８０值为１．５６。提纯病毒经４次免疫家兔,所得抗血清经试管沉淀测定效价为１∶４０９６,琼脂糖双扩散效价为１∶１０２４。     

Specific biological effects of an anti-rat PMN antiserum intraperitoneally infected into f344/n rats

Neutropenia can be produced with antimitotic chemicals, but this method lacks specificity. An alternative is to use antibody-dependent cytotoxicity to produce neutropenia; however, this method has not been completely evaluated with respect to efficacy, specificity, and potential collateral damage, especially to constituents of bone marrow. This study used in vitro and in vivo methods to evaluate specific biological effects of a commercially available rabbit anti-rat neutrophil (PMN) antiserum in F344/N rats. The viability of rat pulmonary alveolar macrophages (PAMs), PMNs, and lymphocytes in vitro was quantified using a trypan blue dye exclusion test. Amounts of antiserum in vitro that rendered PMNs 100% nonviable did not decrease the viability or phagocytic ability of the PAMs and did not decrease the viability of the lymphocytes. Intraperitoneal (IP) injection of the antiserum into rats resulted in complete depletion of the PMNs and about a 50% depletion of the lymphocytes in circulating blood within 24 hours. The numbers of both cell types remained lowered for 5 days, but returned to control values by Day 6 after the IP injection. The antiserum had no effect on the numbers of PAMs or lymphocytes in the pulmonary alveolar airspaces, as determined by quantifying the numbers of these cell types in bronchoalveolar lavage fluid (BALF). The numbers of PMNs in BALF, however, decreased on Days 3 and 4 after IP injection of antiserum, but were not different from control values by Day 5. The viability of the PAMs in BALF of treated rats was not different from control values at any time point. There were no morphological indications that the injected antiserum damaged lung tissue or stem cells in bone marrow. Results demonstrate that the anti-rat PMN antiserum administered IP to F344/N rats depletes circulating PMNs and partially depletes lymphocytes for a period of about 6 days without adversely affecting the precursors of red or white blood cells in bone marrow. We concluded that the antiserum is a relatively specific way to temporarily render rats neutropenic without damaging precursor cells in bone marrow.     

不同动物高免血清对猪肺炎支原体的代谢抑制作用研究

选用猪、兔、鸡、小鼠和豚鼠5种试验动物分别制备猪肺炎支原体高免阳性血清,并测定了其对猪肺炎支原体的代谢抑制效价。再从兔高免血清中纯化特异性抗体,比较抗体纯化前后的代谢抑制效价是否发生变化。结果表明,5种试验动物高免血清对猪肺炎支原体的代谢抑制价分别为0、10~4、10~3、10~5、10~2,纯化后的抗体仍可抑制猪肺炎支原体的生长,代谢抑制价没有明显变化。由此可见,不同动物来源的高免血清对猪肺炎支原体的代谢抑制价不同,代谢抑制作用的主要作用物质为特异性抗体;首次证明病原靶动物——猪的高免抗血清对猪肺炎支原体没有代谢抑制作用,其他支原体是否也表现为对靶动物(或人)抗血清的耐受性还有待进一步研究。     

大麦条纹花叶病毒中国株CP基因克隆分析、原核表达及抗血清制备

 首次用PCR方法,自大麦条纹花叶病毒中国株(BSMV-CH)的RNA₂(GenBank登录号为:AY789694,未报道)中扩增出外壳蛋白(coat protein,CP)基因,并亚克隆到pMD18-T载体上进行测序分析。结果表明BSMV-CH的CP全长597bp,它与BSMV其它株系CP的核苷酸同源性和氨基酸同源性分别为93.1%~93.8%和91.4%~92.4%;与同属的早熟禾半潜病毒(PSLV)和剪秋罗环斑病毒(LRSV)的核苷酸同源性分别为53.0%和41.8%,氨基酸同源性分别为48.1%和40.0%。根据大麦病毒属病毒已经报道的CP氨基酸序列绘制系统发育树,分析表明分离的各毒株在进化上存在明显地理位置的相关性。把CP基因亚克隆到GST融合表达载体pEGX-6p-1上,优化IPTG诱导终浓度后,在37℃ IPTG终浓度为1.0mmol/L时,融合蛋白GST-CP在大肠杆菌BL21中得到了特异表达。12% SDS-PAGE表明,表达产物大小为48.5kDa,主要以包含体形式存在。对蛋白进行定量后,用冰冷的KCl显色,分离表达的蛋白质特异条带制备抗原,免疫家兔得到抗血清,酶联法(enzyme-linked immunosorbant assay,ELISA)测定的抗血清效价为1/6 400。用抗血清对感病大麦和健康大麦进行检测,结果表明抗血清有很高特异性。