伊氏锥虫抗原变异型及其优势代表变异体的分离鉴定

以连续克隆分离的方法,从1株水牛伊氏锥虫中分离到40个克隆群体,从中分离鉴定出18个抗原变异型。经间接免疫荧光试验检测,发现其中2个抗原变异型（即HbTatl.18和HbTatl．15）分别能与约80％和60％克隆群体的血清发生较强的阳性反应,初步确定这2个抗原变异型为该虫株的优势代表变异体。这为伊氏锥虫免疫预防、免疫诊断、分子诊断以及遗传进化等的研究奠定了良好的基础。     

魏氏梭菌毒素疫苗研制及其免疫家兔抗体消长规律

从山东省莱芜、泰安、潍坊等3个养兔场发生疑似梭菌性下痢的病兔体内分离到3株魏氏梭菌，鉴定为A型。利用该分离菌株和A型魏氏梭菌标准株(CVCC37)所产外毒素经甲醛灭活并加入氢氧化铝胶佐荆吸附浓缩后制备了A型魏氏梭菌类毒素疫苗。对该疫苗分别进行了安全性检验、有效免疫剂量试验和接种家兔1～23周血清中抗毒素(Ab)消长规律研究。结果表明，魏氏梭菌外毒素用0．3％甲醛32h能够彻底灭活，并最大可能地保持其抗原性；制备的魏氏梭菌类毒素疫苗无毒副作用，安全可靠；用野毒株和标准株制备的类毒素疫苗对家兔的有效免疫剂量为2mL／只，免疫保护效果可靠；采用野毒株和标准株制备的类毒素疫苗免疫接种家兔后第2周血清抗毒素效价迅速升高，到第4周达最高峰，分别为6．6 log2和7．125 log2，较高抗体滴度维持约17周后缓慢下降，至23周时血清平均抗毒素滴度仍维持在4．0 log2和3．4 log2以上，所以，该类毒素疫苗的免疫保护期可以设定为6个月。     

鹿源坏死梭杆菌毒力菌株FN(AB)94抗原的免疫原性

将坏死梭杆菌分离株 FN(AB) 94厌氧培养后 ,裂解制备抗原 ,在抗原悬液中加入等量福氏完全佐剂 ,配制成乳化抗原 ,以此分别接种 3组 9只家兔 ,并设立对照组。初次免疫后 7d进行第 2次免疫 ,每隔 1周采血 ,检测免疫兔血清中抗体滴度 ,2 8d后免疫兔分别用 2 m L 的 10 8个菌 /m L(2个 ML D)坏死梭杆菌分离株 FN(AB) 94感染 ,3只对照家兔同时感染相同剂量 ;逐日观察攻毒家兔的变化。结果 ,3只对照家兔于感染后的 12～ 2 1d死亡 ;而 9只免疫兔完全能抵抗毒力菌株的感染 ,6个月后仍存活。试验表明 ,坏死梭杆菌分离株 FN(AB) 94裂解抗原加佐剂后 ,具有良好的免疫原性。     

锥虫不同分离株克隆及其等电聚焦分析

将两个伊氏锥虫及一个马媾疫锥虫的不同分离株分别接种用球磷酰胺处理的小鼠，获得五个克隆，用等电聚焦比较其蛋白质差异。结果表明，三个分离株之间有明显区别；马媾疫锥虫显著不同于伊氏锥虫；分离株不同克隆间区别较小，其中广东水牛株两克隆间带型相同，安徽水牛两克隆间带型略有区别。证明伊氏锥虫克隆间变异较小，锥虫不同分离株间变异较大，马媾疫锥虫与伊氏锥虫之间变异最大。     

伊氏锥虫对安锥赛抗药性的分子基础初探

为了探讨伊氏锥虫对安锥赛抗药性的分子基础，将伊氏锥虫单虫克隆，一部分克隆虫通过28只免疫抑制小白鼠，用安锥赛亚治疗剂量治疗，使其产生抗药性，为抗药锥虫；另一部分克隆锥虫也通过28只免疫抑制小白鼠,，但不用药物治疗，为敏感锥虫，即对照，用体外生长抑制法测得抗药和敏感锥虫的IC50分别为43．8和0．16878μg/ml。分别提取它们的总RNA和mRNA，总RNA分离出3条带，即从电泳的阴极到阳棚分别为26s,21s和5s,mRNA主要分布在21sRNA之后。用抑制性消减杂交法，以敏感锥虫的cDNA为试验组（Tester)，抗药锥虫的cDNA为驱动组（Driver)，共挑选出34个阳性克隆，以cDNA来合成探针，用Southern dot blot鉴定，其中18个片段所在的基因是由于抗药性产生而表达量降低，这18个片段长度为300－700bp。     

家畜伊氏锥虫灭活苗制备及免疫研究观察

4种佐剂制备的伊氏锥虫灭活苗，通过小鼠免疫筛选试验，发现Ⅳ号佐剂苗安全性好，免疫原性强，ES抗原在免疫保护中起重要作用。用Ⅳ号佐剂制备的湖北、江苏、广西株伊氏锥虫灭活苗，通过小鼠和家兔2次免疫，对同株虫体的攻击小鼠分别获得20／20、12／12和20／20的保护，并耐受第2、第3次攻击，免疫期长达3－5个月以上。家兔获得7／8保护，体重明显增加，中和凝集抗体效价1：10－80倍。对照小鼠、家兔全部发病死亡。     

伊氏锥虫单一或混合抗原免疫小鼠保护性试验

采用广东克隆（广东水牛伊氏锥虫单虫克隆）、安徽克隆１（安徽水牛伊氏锥虫单虫克隆）、安徽克隆２、新疆克隆（新疆骆驼伊氏锥虫单虫克隆）作单一抗原或混合抗原免疫小鼠试验。用安徽克隆１和新疆克隆单一抗原免疫小鼠，再用相应同株克隆攻击，其保护率在８０％以上，而用异株克隆攻击（安徽克隆１、新疆克隆或广东克隆），保护率低于１７％，表明安徽、新疆和广东伊氏锥虫各株间免疫原性存在差异。用新疆克隆抗原和安徽克隆２抗原混合免疫，再用新疆克隆和安徽克隆２攻击，保护率在８０％以上，用新疆克隆抗原和广东克隆抗原混合免疫，再用新疆克隆和广东克隆攻击，保护率亦在８０％以上，且在免疫后１～２个月仍有效。对新疆克隆抗原和广东克隆抗原混合免疫小鼠脾细胞作体外增殖试验，用不同抗原按５００ｍｇ／Ｌ和１２５ｍｇ／Ｌ高低２种剂量刺激，均产生明显增殖，表明各锥虫抗原存在免疫交叉，但在高剂量组，用同株克隆的刺激增殖效果（ＳＩ）高于异株克隆。     

魏氏梭菌类毒素疫苗研制及其免疫家兔抗体消长规律

本研究分别从山东省的莱芜、泰安、潍坊的三个养兔场发生疑似梭菌性下痢的病兔体分离到三株魏氏梭菌，鉴定为A型。利用该分离菌株和A型魏氏梭菌标准株（CVCC37）所产外毒素经甲醛灭活并加入氢氧化铝胶佐剂吸附浓缩后制备了A型魏氏梭菌类毒素疫苗。对该疫苗分别进行了安全性检验、有效免疫剂量试验和接种家兔1～23周血清中抗毒素（Ab）消长规律研究。结果表明，魏氏梭菌外毒素用03％甲醛32h能够彻底灭活，并最大可能地保持其抗原性；制备的魏氏梭菌类毒素疫苗无毒副作用，安全可靠；用野毒株和标准株制备的类毒素疫苗对家兔的有效免疫剂量为2mL／只，免疫保护效果可靠；采用野毒株和标准株制备的类毒素疫苗接种家兔后第二周血清抗毒素效价迅速升高，到第四周达最高峰。分别为6．6一和7．125log2，较高抗体滴度维持约17周后缓慢下降，至23周时血清平均抗毒素滴度仍维持在4．0-和3．4log2以上，所以，该类毒素疫苗的免疫保护期可以设定为6个月。     

同一克隆两个不同变异体的伊氏锥虫在兔体内抗原变异研究

为了解伊氏锥虫在兔体内感染后期抗原变异的情况及较同一克隆不同变异体锥虫在兔体内抗原变异情况,用伊氏锥虫安徽株单虫克隆ＳｈＴａｔ１．２感染兔,在兔体３０天中每隔３天及感染后５１、５４、５７天（兔５８天死亡）分离兔血中锥虫并克隆,获得１３个克隆锥虫群体,经间接免疫荧光试验和ＡＢＣ酶标记试验鉴定为１０个抗原性互不相同的抗原变异体（Ｖａｒｉａｂｌｅ Ａｎｔｉｇｅｎ Ｔｙｐｅ,ＶＡＴ）,其中早期（感染锥虫３     

伊氏锥虫在小鼠体内抗原变异规律及免疫保护试验

为了探索伊氏锥虫抗原的变异规律及其用于免疫预防的可能性，首先对伊氏锥虫安微株单虫克隆2个早期变异体ShTatl.1、ShTat1.2采用蛋白酶抑制剂TLCK处理，分离纯化这2种变异体的VSG，用ShTat1.1 VSG免疫KM小鼠，ShTat1.1锥虫攻击免疫鼠后6d分离锥虫，经间接免疫荧光试验（IFT）和班点酶标记试验（Dot-EIA)鉴定为ShTat1.2，说明同一克隆锥虫感染兔，小鼠第1次发生抗原变异后产生的变异体相同。依据这一规律，设计了免疫预防保护试验，试验小鼠分3组：一组为未免疫对照组，一组为ShTat1.1VSG单一抗原免疫组，另一组为ShTat1.1 VSG,ShTat1.2 VSG混合免疫抗原免疫组，各组均以ShTat1.1锥虫攻击，结果ShTat1.1 VSG ShTat1.2VSG免疫组小鼠全部获得保护，单用ShTat1.1 VSG免疫组小鼠和未免疫小鼠血中全部出虫、死亡，前者比后者出虫时间、存活时间延长。提示运用伊氏锥虫抗原变异这一规律设计的这种复合抗原，能激发宿主克服虫体抗原变异对免疫保护的干扰。     

H1N1猪流感病毒血凝素蛋白抗原表位的筛选及其抗原性分析

本研究旨在对H1N1猪流感病毒血凝素蛋白抗原分子的模拟表位进行分离鉴定并分析其抗原性。用抗H1N1猪流感病毒血凝素蛋白小鼠血清IgG对噬菌体随机12肽库进行筛选,3轮亲和筛选后,特异性噬菌体得到了有效富集;对随机挑选的47个噬菌体克隆用ELISA进行鉴定,其中40个为阳性;对40个阳性克隆测序得到3种不同氨基酸序列;用Western blotting对获得的3个不同序列的噬菌体克隆进行抗原性分析,显示这3种噬菌体插入短肽能和H1N1猪流感病毒感染小鼠血清特异性结合。结果表明,本试验获得了3种H1N1猪流感病毒血凝素蛋白模拟抗原表位,它们都具有明显的抗原性,为H1N1猪流感病毒的疫苗研究和诊断奠定了基础。     

Antigenic heterogeneity of sialodacryoadenitis virus isolates.

Seventeen field isolates of sialodacryoadenitis (SDA) virus had been isolated from the lung of rats with clinical SDA during epizootics of SDA from 1976 to 1978 in the research laboratory. Based on their neutralization patterns against antisera to strains 681, 930-10, Lu-3, Lu-4 and Lu-7, these isolates were divided into 3 antigenic groups. The first group consisted of 8 isolates which were neutralized by 4 out of 5 antisera at high serum dilution. The second group consisted of 6 isolates which were neutralized by only 2 antisera at high serum dilution. The third group exhibited intermediate neutralization pattern and 3 isolates belonged to it. Considering the time course of virus isolation, it was concluded that three antigenically different SDA viruses had been spread irregularly and ocassionally two had been spread simultaneously in an animal house of rats during the several epizootics of SDA.     

Antigenic change in feline calicivirus during persistent infection.

To determine if antigenic variation occurred during persistent infection of cats with feline caliciviruses (FCV), nine persistent (progeny) isolates from nine different carrier cats were compared antigenically to the original infecting parent strain, FCV 255, by two-way cross-neutralization tests with rabbit antisera. Five of the nine progeny viruses isolated 35 to 169 days after initial infection were antigenically different from the parent strain. These five isolates represented four distinct antigenic phenotypes. The emergence of four distinctly different antigenic variants from a single parent strain indicates that FCV, like many other RNA viruses, exhibits considerable antigenic heterogeneity during replication in its natural host, and supports the hypothesis that antigenic variation contributes to chronic FCV infection.     

Differences in cloning and sub-cloning success rates in four stocks of Trypanosoma evansi and variation in suramin resistance of the clones

Four Trypanosoma evansi stocks with sensitivity to suramin in mice ranging from 0.05 to 160 mg kg⁻¹ were cloned and sub-cloned and the sensitivity of the clones determined. The results suggest that it is easier to clone and sub-clone trypanosome stocks which are sensitive to suramin than those that are resistant to the action of the drug. The clones obtained from the four stocks had sensitivities to suramin which were similar to or different from the parent stocks. These results are important in view of the development of resistance for, in the presence of suramin, these resistant yet heterogeneous populations would provide the material from which selective processes could operate. These observations also suggest that the maintenance and spread of suramin-resistant trypanosomes might be curtailed by their comparative inability to establish themselves in a new host.     

1型鸭甲肝病毒LY0801株VP1基因在鸭胚传代中的变异规律研究

为了解1型鸭甲肝病毒（Duck hepatitis A virus type 1）VP1基因在鸭胚传代中的变异规律,本研究将LY0801株DHAV-1在鸭胚体内传代至30代,分别对1~5、10、15、20、25、30代病毒进行VP1基因的克隆测序。各代次病毒分别以108copies/胚接种9日龄健康鸭胚,测定各组鸭胚的平均死亡时间和病毒在鸭胚尿囊液中的增殖拷贝数。结果表明：DHAV-1在鸭胚传代过程中,不同代次之间出现12个氨基酸的反复变异和同步变异,分别为R43M（K）、T48A、T101S、L169F、T180I、S181L、R183Q、E184A（K）、G187D、D193N、M213R、H219Y;在传代过程中,病毒致死鸭胚的时间逐渐延迟,但病毒在鸭胚内的增殖拷贝数未呈现稳定增长的趋势。     

Absence of host proteins from the surface of Trypanosoma vivax of cattle

Trypanosoma vivax (EATRO 1721) organisms were isolated by DEAE-cellulose chromatography from blood of an experimentally infected calf. Attempts to agglutinate the purified trypanosomes with a rabbit antiserum against whole bovine serum or antisera monospecific for bovine IgG1, IgG2, IgM, complement component C3 or albumin were unsuccessful. The trypanosomes, however, were agglutinated by immune sera of four different calves chronically infected with T. vivax (EATRO 1721). It was concluded that T. vivax organisms purified by DEAE-cellulose chromatography from blood of cattle do not have bovine serum proteins on their surface.     

基于CRISPR/Cas9技术的Wip1基因敲除ST细胞的建立

旨在采用CRISPR/Cas9编辑系统获得Wip1基因敲除的猪睾丸（swine testis,ST）细胞,为后续在细胞水平上探究Wip1基因对ST细胞增殖、凋亡的影响奠定基础。本研究将实验室已有的靶向Wip1基因第一外显子的两条sgRNA（sgWip1-1和sgWip1-2）分别连接到pX330-GFP和pX330-RFP载体,然后共转染ST细胞（从80~90日龄的猪胚胎睾丸组织中分离出未成熟的支持细胞）。利用流式细胞仪收集红绿双荧光阳性的ST细胞,以用于单克隆细胞挑选。对得到的30个单克隆细胞进行基因型鉴定,并将PCR产物进行T-A克隆。结果显示,有29个单克隆细胞出现了基因片段敲除,Wip1基因的片段敲除效率为96.7%。其中单等位基因片段敲除的有5个,纯合双等位基因片段敲除的有8个,杂合双等位基因片段敲除的有16个。本研究高效地构建了Wip1基因敲除的ST细胞,不仅为后续研究Wip1基因对ST细胞增殖、凋亡的影响提供了细胞模型,也为研究Wip1基因对公猪繁殖性能的影响奠定了基础。     

Characterization of monoclonal antibodies against fowl poxvirus

Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.     

Comparison of the ability of feline calicivirus (FCV) vaccines to neutralise a panel of current UK FCV isolates

Feline calicivirus (FCV) comprises a large number of strains which are related antigenically to varying degrees. The antigenic variability creates problems for choosing antigens to include in vaccines. Historically, these have been selected for use based on their cross-reactivity with a high proportion of field strains. However, it is important to determine the current level of cross-reactivity of vaccines and whether or not this may be decreasing owing to widespread vaccine use. In this in vitro study, we have compared the ability of antisera to two vaccine viruses (FCV strain F9 and FCV strain 255) to neutralise a panel of 40 recent UK field isolates. These 40 isolates were obtained by randomised, cross-sectional sampling of veterinary practices in different geographical regions of the UK so as to ensure they were representative of viruses circulating in the veterinary-visiting population of cats in the UK. Virus neutralisation assays showed that both vaccine strains are still broadly cross-reactive, with F9 antiserum neutralising 87.5% and 255 antiserum 75% of isolates tested with antiserum dilutions of 1 in 2 or greater. However, when antibody units were used, in order to take account of differences in homologous titres between antisera, fewer isolates were neutralised, with F9 antiserum showing a slightly higher proportion of isolates neutralised than 255. Multivariable analysis of the sample population of 1206 cats from which the 40 isolates were derived found that vaccinated cats were at a decreased risk of being positive for FCV, whereas cats from households with more than one cat, and cats with mouth ulcers were at increased risk. In addition as cats became older their risk of shedding FCV decreased.     

Trypanosomiasis in different breeds of cattle from Benin.

Blood of different breeds of cattle, namely Lagune from the Atlantic province, Borgou and Borgou x Zebu from the Borgou province, and Somba and Zebu from the Atacora province of Benin, were examined for trypanosome infection. Thick and thin blood smears for trypanosomes, the card agglutination test (CATT), indirect immunofluorescent antibody test (IFAT) and trypanolytic test for antibodies to trypanosomes were used. Trypanosomes were detected in 19.3% (range 9.8-31.4%) of animals by examination of blood smears; antibodies to trypanosomes were found in 89.8% (range 88.4-100%) of samples by IFAT, 50.6% (range 34-87.5%) by CATT and 3.4% (range 1.1-7.1%) by trypanolytic test. Trypanosoma vivax and Trypanosoma congolense were the main species in Benin with a low number of Trypanosoma brucei. Zebu had lower infection rates than trypanotolerant breeds of Benin. The infection rates of various trypanotolerant breeds were not significantly different.