运用伊氏锥虫抗原变异规律进行免疫的初步研究

为了克服锥虫抗原变异对宿主免疫反应的干扰，探索伊氏锥虫病高效保护性抗原，根据伊氏锥虫在宿主体内第一次发生变异产生的变异抗原免疫原性相同的规律，设计了伊氏锥虫变异前抗原（VSG1）和第二次变异所产生的抗原（VSG2）复合物作免疫原，对小鼠进行免疫与单用变异前抗原免疫鼠相比较，两组鼠都用带变异前抗原的虫攻击，另以未免疫的鼠作对照，结果VSG1＋VSG2免疫组小鼠8只全部获得保护，单用VSG1免疫组小鼠10只和未免疫组小鼠10只血中全部出虫而死亡，前者比后者出虫时间和存活时间延长，提示运用伊氏锥虫抗原变异这一规律设计的这种免疫复合物，能激收缩主克服虫体抗原变异对免疫保护的干扰。     

伊氏锥虫单虫克隆的2个变异体的VSG的交叉免疫保护试验

为了考察同一克隆锥虫的不同变异体的可变糖蛋白分子 (VSG)有无交叉免疫作用 ,首先采用蛋白酶抑制剂TLCK处理 ,分离纯化伊氏锥虫安徽株单虫克隆 2个早期变异体ShTat1 1、ShTat1 2的VSG ,然后采用这 2种VSG进行交叉免疫保护试验。试验鼠分为对照组 ,ShTat1 1VSG免疫组 ,ShTat1 2VSG免疫组。用弗氏佐剂按常规进行 3次免疫注射 ,每次抗原用量均为 50 μg/只。三免后第 1 2天 ,将各组小白鼠随机分为 2个小组 ,分别用ShTat1 1、ShTat1 2攻击 ,每只攻虫 1 50 0条。结果攻虫后的对照鼠及异源攻虫后的免疫鼠尾血最早出虫时间均为攻虫后第 3天 ;同源锥虫攻击后免疫鼠的尾血最早出虫时间为第 6天。对照组经攻虫 ,保护率均为 0 /8;免疫鼠经交叉攻虫 ,保护率均为 0 /8;VSG免疫鼠用同源锥虫攻击 ,保护率分别为 5/1 0和 3/1 0。表明各VSG激发的免疫只能抗同源锥虫的感染 ,对异源锥虫感染无保护力     

伊氏锥虫不同株的表面变异糖蛋白提取和重要特性比较

抗原变异是锥虫的重要特征，主要表现于锥虫表面变异糖蛋白（VariableSurfaceGlycoprotein，VSG）的变化，而且锥虫的VSG变异非常频繁，国外对布氏锥虫的VSG研究较多，对中国伊氏锥虫的VSG，杨汉春［1］、李国清等［2］曾作过分离和生化分析，但对中国不同地理宿主株VSG的分离比较尚无报道。而我国北方新疆骆驼伊氏锥虫和南方牛、马伊氏锥虫存在来源、地理、宿主和生化特性的变异[3,4]，我们对这两个虫株的VSG进行了分离、纯化和特性比较研究，以期为伊氏锥虫的抗原变异和伊氏锥虫病的防治研究提供依据。1材料与方法1．1材料和试剂…     

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。     

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。     

接种泰氏锥虫及其抗原使家兔和小鼠产生抗伊氏锥虫的交叉免疫力

用培养的泰氏锥虫制备的死、活两种虫体抗原,接种于家兔和小鼠,一定时间后用伊氏锥虫强毒株攻击,观察其血清抗体的变化及交叉免疫保护能力。试验表明,泰氏锥虫虫体抗原可刺激机体产生较高的体液抗体;免疫动物有一定的抵御伊氏锥虫攻击的能力,与对照组相比,免疫动物血液中虫体出现的时间较迟,最初的数量少,临床症状轻。死虫抗原组的交叉免疫保护能力较活虫抗原组强。     

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     

不同地理株伊氏锥虫灭活苗交叉免疫保护及免疫方法研究

用伊氏锥虫湖北株和广西株制备纯虫灭活苗和含血灭活苗,经小鼠１次、２次免疫,再经强毒伊氏锥虫湖北株、广西株和江苏株交叉攻击。结果湖北株纯虫灭活苗２次免疫鼠,对同株（湖北株）获得３０／３０保护,对异株即广西株和江苏株分别获０／１０和７／１０保护,该苗一次免疫鼠对同株仅获得５／１０保护。广西株纯虫灭活苗２次免疫鼠对同株（广西株）获２０／２０保护,对异株即湖北株和江苏株分别获得０／１０和４／１０保护。含血湖北株伊氏锥虫灭活苗对同株无保护作用,１３只免疫小鼠全部死亡,对江苏株（异株）为９／１０死亡。对照小鼠全部发病死亡。交叉免疫保护试验结果说明,纯虫灭活苗经两次免疫后对伺株虫体产生坚强的免疫保护作用,免疫保护率达１００％,不同地理株伊氏锥虫因抗原变异现象出现不同的免疫保护作用。两次免疫接种小鼠其免疫保护效果比一次免疫的效果好。疫苗中如含有大量非特异物质,虫苗的免疫保护作用将完全丧失。稳定试验表明,灭活苗在室温下保存１个月,在４℃、－２０℃下保存６个月,免疫效果不变。     

伊氏锥虫新疆株在动物体内抗原变异程序的观察

为了探明伊氏锥虫在动物体内抗原变异,以伊氏锥虫新疆株克隆感染兔,每３天采兔血１次,共采血５次,并用环磷酰胺处理的小白鼠对各分离期锥虫予以克隆,获得５个锥虫克隆群体,经ＡＢＣ－酶标试验和间接免疫荧光试验,对各克隆锥虫抗原进行鉴定,结果：获得５个表面抗原性互不相同的克隆群体,说明伊氏锥虫新疆株第１次抗原变异发生在４－６日内,以后每３天变异１次,本研究为进一步探明伊氏锥虫抗原变异规律打下了基础。     

多价伊氏锥虫灭活疫苗制备及免疫保护试验

用湖北株、广西株和江苏株伊氏锥虫经体外培养增殖，再等量混合，制备多价伊氏锥虫灭活苗。疫苗免疫接种小鼠后，再分别用强毒湖北株、广西株和江苏株伊氏锥虫攻击，结果3组小鼠均获12／12保护。免疫保护鼠于免疫后105d时，再作第二次攻虫，结果湖北、广西和江苏组分别获得1／5，3／5，4／5保护，对照组小鼠均100％发病死亡。结果表明多价伊氏锥虫灭活苗对多种虫株均产生了良好免疫保护作用，免疫期达3个月，从而为大面积推广伊氏锥虫病苗提供了有效途径。     

克隆伊氏锥虫不同VAT的某些生物学特性研究

对伊氏锥虫安徽株单虫克隆ＳｈＴａｔｌ感染兔体后３、６、９和１８ｄ所分离的４个抗原变异体ＳｈＴａｔｌ．１、ＳｈＴａｔｌ．２、ＳｈＴａｔｌ．３和ＳｈＴａｔｌ．５的生长特征、对人血清敏感性和对小白鼠致病力三个方面进行了比较研究。结果表明不同抗原变异体的生物学特性不完全一致,它提示伊氏锥虫的抗原变异可能伴有虫体其它生理生化特性的改变。     

克隆伊氏锥虫2个VSG的分离纯化和电泳分析

用蛋白酶抑制剂ＴＬＣＫ对伊氏锥虫安徽株单虫克隆的２个抗原变异体ＳｈＴａｔ１．３和ＳｈＴａｔ１．５的变异表面糖蛋白（ＶＳＧ）进行了分离纯化，采用ＳＤＳ－ＰＡＧＥ和等电聚焦电泳对ＶＳＧ的分子量和等电点进行比较研究。结果，ＶＳＧ的分子量约为４００００，等电点约为ｐＨ５．０，但２个抗原变异体的ＶＳＧ在分子量和等电点都有差异，提示其抗原变异由ＶＳＧ变化引起。     

PCR amplification of RoTat 1.2 VSG gene in Trypanosoma evansi isolates in Kenya

A direct card agglutination test for Trypanosoma evansi, CATT/T. evansi based on the predominant variable antigen-type (pVAT) RoTat 1.2 was evaluated previously in the field in Isiolo District, Kenya. Sixteen out of 51 (31.4%) parasitologically positive camels were negative by the antibody detection test. In the present study, trypanosomes isolated from the camels were analysed in an attempt to determine the cause of the false negative results of CATT/T. evansi. A total of 20 field isolates comprised 16 stocks from camels that were negative by CATT/T. evansi, and 4 from CATT/T. evansi-positive camels. In addition, 15 known T. evansi and four T. brucei were used as reference. Purified DNA samples were tested using an established RoTat 1.2-based polymerase chain reaction (PCR) that yields a 488 bp product for the specific detection of T. evansi. Antibodies to RoTat 1.2 variant surface glycoprotein (VSG) were used in Western blotting to detect RoTat 1.2 VSG linear epitopes. Results of PCR and Western blot showed that the 16 stocks isolated from CATT/T. evansi-negative camels fell into three groups. In Group 1, both the RoTat 1.2 VSG gene and the VSG were absent in three stocks. In five trypanosome stocks in Group 2, the RoTat 1.2 VSG gene was detected, but Western blot was negative indicating absence of the expressed VSG. Five other stocks containing the RoTat 1.2 VSG gene were also in this group. The RoTat 1.2 VSG gene was detected and Western blot was positive in all four trypanosome stocks in Group 3. All four stocks from CATT/T. evansi-positive camels contained the RoTat 1.2 VSG gene and the expressed VSG. The reference T. evansi KETRI 2479 lacked the RoTat 1.2 VSG gene and there was no immune reactivity detected by Western blot. The rest of the reference T. evansi stocks examined contained the RoTat 1.2 VSG gene. All the four T. brucei samples examined were negative by PCR and Western blot. In conclusion, this study showed that the RoTat 1.2 VSG gene was absent from some T. evansi trypanosomes in Kenya.     

Expressed truncated N-terminal variable surface glycoprotein (VSG) of Trypanosoma evansi in E. coli exhibits immuno-reactivity

The variant surface glycoprotein (VSG) of trypanosome is an important part of its body surface coat, which is expressed in early, middle and late stages of infection contributing a major diagnostic value. In the present study, the 5' end of the partial VSG gene sequences (681 bp) encoding N-terminal protein of RoTat 1.2 VSG (227 amino acid) was amplified, cloned into pET32a vector, and expressed in prokaryotic system. The fused His-tagged expressed VSG protein (43 kDa) of the Trypanosoma evansi was characterized in SDS-PAGE and immunoblotting using hyperimmune/immune sera raised against buffalo, dog, lion and leopard isolates of T. evansi. The expressed protein remained immunoreactive with all the sera combinations. The animals immunized with whole cell lysate or recombinant protein showed similar antibody reactions in ELISA and CATT (Card Agglutination Test for Trypanosomiasis). This study suggests the expressed recombinant truncated VSG is having its importance for its possible use in sero-diagnosis of surra.     

General expression of RoTat 1.2 variable antigen type in Trypanosoma evansi isolates from different origin

The variable surface glycoprotein of Trypanosoma evansi RoTat 1.2 variable antigen type (VAT) is used as an antigen in different antibody detection assays for T. evansi. To obtain more information on the predominant character of RoTat 1.2 and its diagnostic potential in antibody detection tests, we checked its expression in 10 different T. evansi stocks and clones from different parts of the world. Cryostabilates were injected into mice and the trypanosomes of the first peak parasitaemia were screened for the presence of RoTat 1.2 by VAT specific immunofluorescence. To monitor the appearance of RoTat 1.2 specific antibodies during infection, rabbits were infected and serologically tested at different time intervals with VAT specific immune trypanolysis, CATT/T. evansi, LATEX/T. evansi and ELISA/T. evansi.Test results confirm the predominant character of RoTat 1.2.     

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.     

伊氏锥虫变异表面糖蛋白抗原双抗体夹心ELISA检测方法的建立及初步应用

A double antibody sandwich ELISA for detection of Trypanosoma evansi variant surface glucoprotein (VSG) antigen was established by using two monoclonal antibodies against Trypanosoma evansi VSG antigen.The result of sensitivity showed when positive serum was diluted to 3200 doubles, its D_{450 nm} value was still greater than the critical value of positive and negative,and specificity test result showed that there was no cross reaction with the antigens of Theileria,Toxoplasma gondii,Chlamydia and Fasciola gigantica.82 clinical sera were detected by this double antibody sandwich ELISA and its positive rate was 9.76%.The experiment results demonstrated that this double antibody sandwich ELISA for detection of Trypanosoma evansi VSG antigen had good detecting sensitivity and specificity,and could be used as an effective method for clinical detecting buffalo Trypanosoma evansi disease.     

共免疫对跳蚤叮咬引起的猫过敏性皮炎保护效果评价

为评价共免疫r FSA1和p VAX-FSA1对于猫跳蚤过敏性皮炎(FAD)的预防免疫保护效果,用人工合成的跳蚤唾液抗原(FSA1)基因,分别构建原核表达质粒p ET28a-FSA1和真核表达质粒p VAX1-FSA1。p ET28a-FSA1转化E.coli BL21(DE3)感受态细胞,诱导表达纯化FSA1,获得r FSA1。12月龄以上猫共免疫r FSA1+p VAX-FSA1,第14天实施二免后,进行跳蚤叮咬,连续4轮,跳蚤叮咬结束后,统计皮炎评分。结果显示,第4轮攻跳蚤后共免疫r FSA1+p VAX-FSA1组的猫皮炎评分远远低于FAD对照组,说明共免疫r FSA1+p VAX-FSA1能抑制FAD症状,对跳蚤叮咬猫引起的过敏性皮炎有显著保护效果,可以作为预防猫跳蚤过敏性皮炎的良好办法。