RT-PCR法分离伊氏锥虫VSG基因的研究

应用异硫氰酸胍一酚一氯仿一步法分离伊锥虫安微株克隆虫体ＳｈＴａｔｌ的二信抗原变异体ＳｈＴａｔ１．３和ＳｈＴａｔ１．５总ＲＮＡ，在含有甲醛物琼脂糖凝胶电泳后，转印到硝酸纤维膜上。根据布氏锥虫ＶＳＧｍＲＮＡ３’端保守序列，设计合成了一个互补于它的含十八个碱基的寡核苷酸５’－－ＧＧＴＧＴＴＡＡＡＡＴＡＴＣＡＧ－３’，经＾３２Ｐ标记，Ｎｏｔｒｈｅｒｎ杂交，首次证明２伊氏锥虫含有同样的保守序列。利用合成的十     

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     

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。     

伊氏锥虫抗原变异规律

国内外已有众多文章报道了伊氏锥虫表面抗原变异现象的特点，但均较零散，本文将这些报道进行分析综合，从中找出共同点，归纳出伊氏锥虫抗原变异的一些规律，计有伊氏锥虫抗原变异的频率、抗原变异的程序、变异体的数量、优势变异体和非优势变异体，伊氏锥虫感染动物后第一次发生抗原变异的时间和特点等，作者并对尚需继续探索的有关规律性的研究内容也提出了看法。     

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。     

伊氏锥虫同工梅,蛋白质和抗原组分的比较研究

本文采用生化技术对八个中国伊氏锥虫株及一个布氏锥虫株的同工酶、蛋白质的抗原组分进行比较研究。根据同工酶电泳结果，可将伊氏锥虫和与其形成形态上不能区分的布氏锥虫区别开来，亦可将伊锥虫分为两个酶株群（Ｚ１、Ｚ２）。根据ＳＤＳ－聚丙烯酰胺凝胶电泳，等电聚焦电泳和免疫印迹试验的结果，可将酶株群Ｚ１分成五个同多肽群（株）。本研究结果表明：中国伊氏锥虫遗传传 变异程度较低，是一个相对稳定的种群。     

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。     

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。     

应用单克隆抗体检查伊氏锥虫抗原变异

用广西骡株伊氏锥虫可溶性抗原免疫的BALB/C小鼠脾细胞与小鼠骨髓瘤细胞(SP_2/O)融合,获得了在ELISA中能与广西骡株伊氏锥虫可溶性变异表面糖蛋白反应的2个分泌IgG_1的单克隆抗体(McAb)。用McAb于ELISA抑制试验对四份不同期的广西骡株伊氏锥虫阳性血清的抗体成分进行了分析,结果四份锥虫阳性血清对于McAb与相应抗原决定簇的结合均未出现明显的抑制作用。表明血清样本间很少或完全没有与McAb相类似的抗体。这证明伊氏锥虫存在多个血清型亦即变异抗原型。     

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     

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.     

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。     

Induction of protective immune response in mice by a DNA vaccine encoding Trypanosoma evansi beta tubulin gene

Surra, caused by Trypanosoma evansi, is an economically important veterinary disease of the tropics. Lack of effective drugs or vaccines have made surra a severe economic burden particularly in Asia and sub-Saharan Africa. In this study, a naked DNA construct encoding full length T. evansi beta (β) tubulin gene was used to immunize mice, to elicit a T. evansi β tubulin protein specific humoral immune response, delineated by ELISA. The serum cytokine profile post immunization, as determined by flow cytometry bead based assay, showed a predominant T helper cell Type 1 (Th1) response with significant increase in levels of IFNγ and TNFα. Lethal challenge with T. evansi blood-form trypomastigotes post immunization generated a β tubulin specific recall response and a stronger Th1 type serum cytokine profile which correlated with an extended survival and better control of parasitemia in the immunized mice.     

Host parasite relationships in Trypanosoma (duttonella) vivax with special reference to the influence of antigenic variation

A mouse model system was used to study various aspects of host and parasite relationships in Trypanosoma vivax infections. These included the phenomenon of antigenic variation, the variable parasite antigens responsible for this phenomenon, parasite-host adaption, host immune responses and the role of genes in the major histocompatibility complex in the control of infection. While the mouse model system has allowed investigation of these aspects of host parasite relationships, it is clear that the system is much more limited than those generally used in T. brucei spp and T. congolense infections. This is indicated by the discovery that not all VATs of T. vivax were equally infective for mice, though in some cases infectivity could be improved by bovine serum supplementation and/or immunosuppression of the mouse host. In the case of rats, infection was even restricted to a smaller number of the VATs studied. It was, however, possible to biochemically characterize the variable surface antigen carried by T. vivax and show its similarity to those carried by T. brucei and T. congolense. The H-2 complex was found not to influence acquired resistance of inbred strains. Cyclic transmissions of T. vivax infections to goats combined with chemotherapy were carried out in an attempt to induce protection to subsequent infection as has been shown in T. brucei and T. congolense infections. Such protection could, however, not be obtained, The failure of the metacyclic VATs to induce immunity, was perhaps due to rapid decrease in antibody titres to bloodstream VATs found after treatment and prior to rechallenge. The usefulness of the mouse model system in elucidating the mechanisms responsible for the non-H-2 linked differences in susceptibility to T. vivax infections should be further explored and its relevance to mechanisms of trypanotolerance in domestic ruminants defined.