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1.
为评价Serpin基因重组表达产物对预防鸡柔嫩艾美耳球虫(Eimeria tenella,E.tenella)感染的效果,克隆和表达E.tenella Serpin基因,对表达产物进行初步纯化和复性,制备免疫原.设计了重组蛋白肌肉注射组、重组蛋白口服组、重组蛋白滴鼻组3个试验组以及红、白对照组.分别于7、14和28日龄对雏鸡进行3次免疫,35日龄时用3×104个E.tenella孢子化卵囊攻虫,第7天末宰杀,对各组的存活率、相对增重率、卵囊减少率、ACI等指标进行统计分析.结果表明,各免疫组的平均增重与红对照组相比均有显著增加(P＜0.05),其中以重组蛋白肌肉注射组增重最多,相对增重率为66.73％,优于其他免疫组;各免疫组卵囊产量较红对照组均有显著的下降,其中重组蛋白肌肉注射组卵囊减少率最高,为30.01％;各免疫组ACI均明显高于红对照组,其中以重组蛋白口服组最高,但仍低于160,暗示Serpin蛋白并不是一个理想的球虫疫苗保护性抗原.  相似文献   

2.
本研究旨在评价表达柔嫩艾美耳球虫(Eimeria tenella)顶膜抗原1(Apical membrane antigen 1,Et AMA1)基因的DNA疫苗对雏鸡柔嫩艾美耳球虫人工感染的免疫保护效果。将Et AMA1基因和鸡IFN-γ基因插入真核表达载体p CAGGS中,分别构建了真核表达重组质粒p CAGGS-Et AMA1和p CAGGS-Et AMA1-IFN-γ。将重组质粒p CAGGS-Et AMA1转染进293T细胞中,经间接免疫荧光和Western blot实验鉴定,观察其在体外表达情况。将p CAGGS-Et AMA1和p CAGGS-Et AMA1-IFN-γ于7日龄和14日龄腿部肌肉注射免疫雏鸡,21日龄人工感染1×104个柔嫩艾美耳球虫孢子化卵囊,第29日龄时处死试验鸡,以平均增重、卵囊产量和病变记分来评价重组质粒的免疫保护效果。结果显示,p CAGGS-Et AMA1转染的293T细胞出现明显的红色荧光;两种真核表达重组质粒免疫组鸡的平均增重与未攻虫组的平均增重差异不显著,不具有统计学意义,而未免疫攻虫组则与未攻虫组在增重方面差异具有显著统计学意义;免疫组鸡相较于未免疫攻虫组在病变记分方面显著下降;免疫组的卵囊减少率分别为67.43%和72.89%;p CAGGSEt AMA1-IFN-γ组在增重、盲肠病变记分和卵囊减少率方面都优于p CAGGS-Et AMA1组,但差异不具有统计学意义。表明构建的2种重组质粒对雏鸡柔嫩艾美耳球虫感染有一定的免疫保护效果。  相似文献   

3.
将毒害艾美耳球虫重组蛋白rEnApiAP2单独免疫(单免)或分别与柔嫩艾美耳球虫重组蛋白rEtGAM56或rEtGAM59联合免疫(联免)雏鸡,同时设rEtGAM56单免、rEtGAM59单免、未免疫攻虫和空白组为对照,分别攻毒害艾美耳球虫或柔嫩艾美耳球虫,以成活率、排出血便数、平均增重、相对增重率、病变记分、卵囊减少率、抗球虫指数(ACI)和血清抗体水平为指标,评价rEnApiAP2单免及与rEtGAM联免的免疫保护效果。结果显示：各试验组的成活率均为100%;与未免疫攻虫组比较,各免疫组鸡排血便堆数减少、平均增重增加。在rEnApiAP2单免组间,低剂量组的相对增重率(82.36%)、卵囊减少率(73.88%)和抗球虫指数(155.26)均为最高,平均病变记分最低(1.71)。攻毒害艾美耳球虫后,联免组的ACI值均大于相应的单免组,其中rEnApiAP2与rEtGAM56联免组的ACI值(169.83)最高。攻柔嫩艾美耳球虫后,rEnApiAP2单免组的ACI值(128.37)低于rEtGAM56、rEtGAM59单免组(130.12、151.88),rEnApiAP2与rEtGA...  相似文献   

4.
棒状体蛋白(EtRP)在柔嫩艾美耳球虫入侵宿主细胞的过程中起重要作用。该蛋白在球虫发育阶段的早期表达,具有保护性抗原的性质,其相应抗体可抑制球虫对宿主的感染。采用纯化后的p GEX-6P-1-EtRP重组蛋白免疫BALB/c小鼠制备了抗EtRP的单克隆抗体,将不同剂量的单克隆抗体注射试验鸡群再对免疫鸡群攻虫,7 d后致死鸡群并计算卵囊减少率、相对增重率以及盲肠病变评分情况。结果表明,所制备的单克隆抗体亚型为IgG1,经Western-blot检测能特异性识别重组蛋白,经间接ELISA测定其效价大于1∶128 000。此外,单克隆抗体对鸡的被动保护性试验结果显示相对增重率、病变评分、卵囊减少率均随着免疫剂量的增加而增大,表明一定剂量的单克隆抗体可以有效减少柔嫩艾美耳球虫对鸡群的感染。  相似文献   

5.
为评价gain56基因重组表达产物作为亚单位疫苗预防鸡巨型艾美球虫(E.maxima),感染的效果,以E.maxima NT株配子体总RNA为模板,RT.PCR扩增和克隆gain56基因,选择该基因两段丰富抗原表位的编码区片段.利用原核表达载体pGEX-6P-1在大肠杆菌中进行截短表达.以可溶性的GST-gain56-2融合蛋白为免疫原,设立高、中、低(1.0 mg、0.5 mg、0.25 mg)3个剂量,单独或使用弗氏完全佐剂,于5 d、12 d和19 d对雏鸡进行3次免疫,26 d用5 ×10~4个E.maxima孢子化卵囊进行攻虫,8 d后迫杀,对各组的存活率、相对增重率、卵囊减少率、ACI等指标进行统计分析.结果显示:就相对增重率而言各免疫组比未免疫攻毒组的高27.6%以上,而各免疫组之间差异不显著(p>0.05);就卵囊减少率指标而言,各免疫组较未免疫攻毒组均有不同程度减少,但是均小于75%;就ACI指标而言,各免疫组较未免疫攻毒组均有不同程度增加,以中剂量蛋白佐剂组为最高.GST-gam56-2融合蛋白对于E.maxima感染具有一定的免疫保护效力.  相似文献   

6.
利用PGEX-6P-1融合表达系统将SO7基因在大肠杆菌中进行融合表达,对表达产物进行初步纯化和复性,制备免疫原。分别使用不同剂量的人参总皂甙和重组γ-干扰素以及弗氏完全佐剂作为免疫佐剂,于5日龄、12日龄和19日龄对雏鸡进行3次免疫,同时设立蛋白口服免疫组、蛋白皮下注射免疫组、卵囊口服免疫组、未免疫未攻毒组和未免疫攻毒组作对照,于26日龄用105个柔嫩艾美耳球虫卵囊进行攻毒,8d后扑杀,对各组的存活率、相对增重率、病变减少率、相对卵囊产量和抗球虫指数ACI等指标进行统计分析,比较免疫保护效果。与非免疫攻毒组以及不加佐剂的免疫攻毒组相比,佐剂使用后各组的增重、病变记分、相对卵囊产量、ACI等指标均有一定的改善,对柔嫩艾美耳球虫的人工感染可以提供部分保护。3种佐剂中,γ-干扰素和人参总皂甙能增强重组蛋白的免疫保护效果,且佐剂的剂量对免疫保护的效果有一定的影响,以5000Uγ-干扰素效果最佳,效果与E.tenella活卵囊免疫相当,弗氏完全佐剂的免疫增强效果不明显。单独使用重组蛋白皮下注射或口服免疫,虽有一定的保护作用,但不明显。一定剂量的γ-干扰素对SO7抗原的免疫保护效果起明显的增强作用,是一个具有很好临床应用前景的新型佐剂。  相似文献   

7.
为确定使雏鸡获得良好免疫效果的最小巨型艾美耳球虫(E.maxima)早熟株接种量,分别于4日龄对免疫攻毒组以不同剂量进行首免,11日龄以首免2倍剂量对二次免疫攻毒组鸡进行二免。一次或二次免疫后7 d或10 d用同源亲本株进行攻毒,检测免疫攻毒鸡相对增重率、卵囊减少率、病变记分减少率(RLS)和抗球虫指数(ACI)。结果显示,一次免疫剂量≥400个孢子化卵囊/只或两次免疫的一免剂量和二免剂量≥100个孢子化卵囊/只和200个孢子化卵囊/只时,各免疫攻毒鸡的相对增重率≥85%,RLS≥70%,卵囊减少率≥75%,相对保护率≥90%,ACI≥170,均达到良好免疫效果。故将E.maxima早熟株的一次免疫最小免疫剂量定为400个孢子化卵囊/只,两次免疫最小免疫剂量定为一免100个孢子化卵囊/只和二免200个孢子化卵囊/只。  相似文献   

8.
将150只海蓝雏鸡分为5组,每组30只,3日龄首免,2周后加强免疫1次,二免后2周用鸡柔嫩艾美尔球虫攻击.通过测定免疫后鸡只的淋巴细胞转化水平,抗体水平、IL-1诱生活性,比较分析不同剂量的香菇多糖对3-1E重组蛋白免疫原性的增强作用;通过测定攻虫后鸡只存活率、相对增重率、卵囊值和抗球虫指数的变化,分析香菇多糖对3-1E重组融合蛋白免疫保护效果的影响.结果表明,香菇多糖能够显著增强淋巴细胞转化水平(P<0.05)和血清抗体水平(P<0.05),但不能明显提高IL-1的诱生活性(P>0.05);而且能够显著增强3-1E重组蛋白的免疫保护效果.表明香菇多糖对3-1E重组蛋白具有较好的免疫增强作用.  相似文献   

9.
本研究初步评价了柔嫩艾美耳球虫热休克蛋白70(EtHSP70)的免疫保护效果。以RT-PCR方法克隆获得E.tenella广东株的Ethsp70基因序列,插入表达载体pMAL-c2X后转化入大肠杆菌Rosetta株,经IPTG诱导,可高效表达分子量为112 ku的可溶性融合蛋白,表达量约占菌体总蛋白的27%。将Ethsp70基因插入真核载体pcDNA6,构建真核表达质粒pcDNA-Ethsp70。用纯化的重组融合蛋白(rEtHSP70)和pcDNA6-Ethsp70质粒分别以100μg/只剂量肌注免疫雏鸡,攻虫后以盲肠病变计分、相对盲肠卵囊产量(ROP)、相对增重率和抗球虫指数(ACI)为评价指标,结果表明2个免疫组相对盲肠卵囊产量分别为39.4%、45.2%,抗球虫指数由89分别提高至免疫组的164和150。提示EtHSP70可能是一种有潜在应用价值的保护性抗原。  相似文献   

10.
为了确定巨型艾美耳球虫(Eimeria maxima)早熟株合适的免疫剂量,本文设立7个早熟株免疫攻虫组、7个母株免疫攻虫组、1个不免疫攻虫组和1个不免疫不攻虫组,7个早熟株/母株免疫组的免疫剂量为孢子化卵囊100、200、400、600、800、1000和2000个/羽,经嗉囊感染,7日龄首次免疫,14日龄以同等剂量进行第二次免疫,21日龄以8×104个/羽的同源母株进行攻虫,28日龄结束试验,以增重、肠道病变记分和卵囊减少率为试验指标。对早熟株中免疫保护效果较好的2个免疫剂量进行重复试验,免疫方法、试验周期、试验指标和攻虫剂量同第一批试验。结果显示:早熟株和母株200、400、600、800、1000、2000个/羽免疫组攻虫后的增重与健康对照组差异不显著(P>0.05),100个/羽免疫组明显低于健康对照组(P<0.05);各免疫组的卵囊减少率均达90%以上,肠道病变记分均与不免疫攻虫组差异不显著(P>0.05)。用早熟株200、400个/羽进行免疫重复试验,两个免疫组攻虫期间的增重和肠道病变记分均与健康对照组差异不显著(P>0.05),而与不免疫攻虫组差异显著(P<0.05),其卵囊减少率均在86%以上。研究结果表明,该巨型艾美耳球虫早熟株保持了良好的免疫原性,每只鸡免疫200或400个孢子化卵囊均可诱发鸡产生良好的免疫保护力,可考虑以200个孢子化卵囊作为该早熟株在疫苗制备中的推荐免疫剂量。  相似文献   

11.
以重组质粒pGEM-T-gam82为模板,构建巨型艾美耳球虫gam82基因核酸疫苗.采用MTT法与ELISA对该核酸疫苗接种鸡体后淋巴细胞增殖与抗体水平的变化进行检测,结果显示,该核酸疫苗具有一定的免疫原性.  相似文献   

12.
A cDNA library was constructed with Eimeria necatrix merozoite mRNA and immunologically screened by chicken sera against this parasite. One of the positive clones containing an insert of 879 nucleotides, pNP19, showed similarity to part of a published gene expressed in E. tenella merozoite by the homology search system. The inserted DNA was subcloned into baculovirus, and a 35-kD protein was expressed, purified, and used for the antigen in enzyme-linked immunosorbent assay (ELISA). Antibodies from the chickens vaccinated with the E. necatrix attenuated strain, Nn-P125, were detected from 14 days after vaccination by ELISA. The mean absorbance increased rapidly to a peak around 21 days after vaccination; thereafter, it began to decline. Even though some of the vaccinated chickens showed very low levels of antibody response to the recombinant protein 56 days after vaccination, they were protected against challenge with virulent strain of E. necatrix. The mean absorbances in sera from both vaccinated and nonvaccinated chickens highly increased 14 days after challenge. On the other hand, the antibody was not detected in ELISA when chickens were exposed to other Eimeria species such as E. tenella, E. acervulina, and E. maxima. These results demonstrate that this recombinant protein is suitable for detecting the specific antibody in chickens infected with both attenuated and virulent strains of E. necatrix.  相似文献   

13.
A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.  相似文献   

14.
Anticoccidial effects of xanthohumol   总被引：1，自引：0，他引：1  
Allen PC 《Avian diseases》2007,51(1):21-26
Xanthohumol (XN), a prenylated chalcone from the hops flower, was examined for its ability to reduce invasion of Madin-Darby bovine kidney (MDBK) cells by Eimeria tenella sporozoites (SZ), as well as to reduce invasion by E. tenella and E. acervulina SZ in the chick host. Additionally, XN was tested as an anticoccidial feed additive at 20 ppm against challenge infections with E. acervulina, E. maxima, and E. tenella. Cell invasion by E. tenella SZ was inhibited 66% by treatment of SZ with 22 ppm XN. This inhibition was associated with an apparent physical disruption of the apical ends of the SZ. Rectal challenges with E. tenella SZ treated with 5, 10, and 20 ppm XN resulted in significantly reduced gross-lesion scores and normal chick-host weight gains compared with challenge with untreated SZ. Oral challenges with similarly treated E. acervulina SZ, accomplished with prior antacid treatment, resulted in significantly reduced gross lesions and reduced oocyst shedding compared with challenge with untreated SZ and were associated with physical disruption of sporozoite morphology. In a pilot test, provision of feed supplemented with 20 ppm XN for 3 days before challenge to 6 days after challenge did not control challenge infections with E. acervulina, E. maxima, or E. tenella as judged by measurements of weight gain, feed conversion, and gross lesions. Although XN-fed chicks infected with E. acervulina and E. maxima shed fewer oocysts than those on control feed, the differences in numbers were not statistically significant (P > 0.05).  相似文献   

15.
In order to evaluate whether infection with E. canis alters the expression of major histocompatibility complex (MHC) class I and/or MHC class II receptors, and by doing so alters the immune response to the organism, flow cytometry was performed on DH82 cells infected with Ehrlichia canis (90% infection) and on uninfected DH82 cells of the same passage, using anti-canine MHC class I and II antibodies. MHC class II expression was evident in 47.6 and 46.2% (mean 46.9%) of uninfected DH82 cells using two different anti-MHC class II antibodies, while no MHC class II expression was evident in DH82 cells infected with E. canis. The present results indicate that infection of DH82 macrophages with E. canis down-regulates their MHC class II receptors. These results suggest a possible mechanism by which E. canis evades the immune system.  相似文献   

16.
1. Experiments have been carried out to compare weight gain, mortality and oocyst production in 7 inbred and partially inbred lines of chickens after challenge with the coccidial parasites Eimeria tenella and E. maxima. 2. There were large differences between lines in the effects of challenge on weight gain and mortality for both species of parasite. However, the lines suffering the greatest mortality were not those showing the greatest effects on weight gain, indicating that mortality alone is not an adequate criterion in selection for resistance. 3. Although oocyst production differed between lines there was no correlation with mortality or with weight loss, implying that the variation observed in these traits was not due to a restriction of the parasite but to an accommodation of its effects. 4. Mortality and weight loss resulting from challenge with E. tenella in the different lines did not correlate with that caused by E. maxima. 5. There was evidence of an association of genes of the major histocompatibility complex genes with mortality, but not with weight loss or oocyst production: there was no indication of association of resistance to coccidiosis with resistance to Marek's disease.  相似文献   

17.
Donor chickens given feed medicated with one or two levels of decoquinate or given non-medicated feed were infected with oocysts of Eimeria tenella or E. maxima per os. Twelve hours after inoculation with oocysts liver, mid-intestine or ceca homogenates were fed to previously uninfected recipient chickens. The results showed that continuous medication with decoquinate was effective in preventing the transfer of sporozoites from the intestine to the liver. Oocysts were detected in the feces of all recipients of tissue from non-medicated donors, showing that some sporozoites of E. maxima and E. tenella are normally transferred to liver. Young broiler chickens were immunized by oral inoculation of E. maxima oocysts. The immune status of similar chickens inoculated with sporozoites of the same species directly into the liver or spleen were assessed. During the experimental period half of the chicks were provided with non-medicated food and the remainder were given feed supplemented with decoquinate; decoquinate was effective in arresting the development of the sporozoites. Two weeks after initial infection the birds were challenged with oocysts of E. maxima per os. Injection of sporozoites into the spleen did not protect against challenge. Birds inoculated with sporozoites into the liver were unable to develop a significant level of immunity. When the drug pressure was removed from these birds, parasitism of the intestine occurred and immunity developed.  相似文献   

18.
为克隆、表达日本血吸虫假想蛋白SJCHGC068068编码基因（暂命名为Sj06868）并观察重组抗原的免疫预防效果。应用PCR技术从日本血吸虫7 d童虫、14 d童虫mRNA反转录制备的cDNA中克隆到Sj06868基因的46-438 bp DNA片段,BLASTn分析未发现其他物种中的同源性序列,也未发现其保守序列和相关功能结构域。以pET32a为表达载体、Sj06868基因在大肠杆菌Rosetta（DE3）中获得高效表达,表达产物rSj068068分子量为36 kDa,能被感染血吸虫14 d兔血清识别。将纯化的重组抗原与佐剂ESSAI ISA 206 CELL混合后免疫BALB/c小鼠,与佐剂对照组和PBS对照组相比,分别获得了31.63%、29.19%减虫率以及55.63%、56.27%肝脏虫卵减少率。用ELISA检测各组血清中抗rSj068068特异性抗体结果显示,免疫组在攻击感染前、佐剂对照组和PBS对照组在感染后均产生了较高水平的特异性抗体。本研究结果说明,Sj068068是日本血吸虫特有的童虫期高表达基因,rSj068068在抗血吸虫疫苗和血吸虫感染的诊断方面均有潜在应用价值。  相似文献   

19.
提取扬奇青霉总RNA,反转录合成cDNA,PCR扩增α-半乳糖苷酶agl1基因的编码区DNA序列,连接到原核表达载体pET28a(+)的多克隆位点,获得重组表达载体,转化大肠杆菌BL21,通过IPTG诱导获得agl1基因的特异性表达。结果表明:8mol/L尿素变性处理后的重组蛋白,经过Ni2+亲和柱纯化,SDS-PAGE检测该重组蛋白分子量约82ku,与预测结果相符。纯化的酶蛋白依次经过6、4、2mol/L和0mol/L尿素梯度透析复性后,α-半乳糖苷酶酶活为0.4U/mL。  相似文献   

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