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1.
《中国猪业》2012,(3):70-70
典型猪瘟(CSF)由典型猪瘟病毒(CSFV)引起,是一个高度传染的猪病,给猪业生产造成巨大的经济损失。病毒衣壳糖蛋白E(ms)和E2是感染该病毒后诱导产生CSFV抗体的主要靶向目标。毕赤酵母表达E2蛋白(yE2)可产生免疫保护.对抗CSFV攻毒。  相似文献   

2.
以小鼠为动物模型,对此前构建的表达H3N2亚型猪流感病毒(SIV)血凝素(HA)基因的重组伪狂犬病病毒(rPRV-HA)进行了免疫效力评价。按每只10^5.0 TCID50 rPRV-HA的剂量通过滴鼻接种8周龄雌性BALB/c小鼠(n=60),同时设Bartha-K61免疫对照组(n=60)、非免疫攻毒对照组(n=20)和非免疫不攻毒对照组(n=10)。于免疫后不同时间分别从rPRV-HA免疫组和Bartha—K61免疫对照组随机剖杀一定数量的小鼠,其余小鼠于免疫后第28天用10^5.0 TCID50同亚型SIV毒株A/Swine/Heilongjiang/74/2000(H3N2)进行强毒攻击。攻毒后第4、7、14天分别剖杀小鼠,进行间接免疫荧光、病毒分离、血清学和病理组织学检测。结果表明,重组病毒主要分布于肺脏;免疫后14d起,从rPRV—HA免疫组及Bartha—K61免疫对照组均可检测到针对PRV的荧光抗体;从rPRV—HA免疫组可以检测到针对SIV的荧光抗体和血凝抑制抗体,而各对照组均呈阴性。攻毒后从rPRV—HA免疫组小鼠未分离到攻击病毒,血凝抑制抗体显著升高,病理变化显著轻于对照组,表明rPRV—HA免疫小鼠可以抵抗同亚型SIV的攻击,可以作为rPRV—HA免疫效力评价模型。  相似文献   

3.
从广西南宁某猪场分离1株病毒(GX-3/98),通过RT-PCR扩增出猪瘟病毒约250 bp的E2基因主要抗原编码区,与猪瘟石门系强毒株及兔化弱毒株核苷酸序列同源性分别为80.6%和81.1%,属于基因Ⅱ群,subgroup 2.1亚型。该毒株经本动物传3代,均不表现典型的猪瘟临床症状。用猪瘟兔化弱毒疫苗免疫后,以此分离病毒攻毒,进行免疫保护相关试验,结果免疫组100%(2/2)获得保护,且在攻毒前、后扁桃体HCFA检测均为阴性。对照组(非免疫猪)攻毒后50%(1/2)死亡,另1头猪耐过。扁桃体HCFA检测,于攻毒后1周开始出现阳性结果,且一直持续到猪死亡的第79天。本试验结果初步表明,我国现行使用的猪瘟兔化弱毒苗对目前猪瘟病毒流行毒株仍具有良好的保护力。GX-3/98流行株为1株毒力较低的猪瘟病毒,能够长时间散毒。  相似文献   

4.
应用PCR从pMD18-T-E0质粒中扩增编码CSFV E0蛋白的基因片段,定向克隆到重组腺病毒Adeasy-1系统的穿梭质粒pAdTrack-CMV上,采用细菌内同源重组“两步转化法”构建携带CSFV E0基因的重组腺病毒基因组质粒pAdEasy-E0,转染293细胞,成功包装出重组腺病毒pAd-E0,PCR证实E0基因已整合至腺病毒基因组中,用Western blot检测到重组病毒感染293细胞中E0蛋白的表达。重组病毒免疫小鼠和猪,结果2次免疫后产生明显的免疫应答,ELISA检测小鼠血清抗体滴度分别为1∶512和1∶10240;猪血清抗体滴度分别为1∶16和1∶64。本研究成功构建了表达猪瘟病毒E0基因的非复制型重组腺病毒,该重组病毒免疫小鼠可产生较高的抗体滴度,免疫猪后能提供一定的保护效果。  相似文献   

5.
为评价重组腺病毒融合表达猪瘟病毒(CSFV)E0和E2蛋白的免疫保护效果,本研究以CSFV基因组为模板,应用RT-PCR扩增E0和E2蛋白的编码基因,通过pET-32a载体将E0和E2基因串联,形成pET-E0-E2重组质粒。用KpnⅠ和NotⅠ双酶切pET-E0-E2得到E0-E2融合基因,定向亚克隆于穿梭载体pAdTrack-CMV中,采用"两步转化法"在细菌内同源重组,构建携带E0-E2基因的重组腺病毒转移载体质粒pAdEasy-E0-E2,经PacⅠ酶切线性化后转染人胚胎肾细胞(HEK293),成功包装出重组腺病毒(rAd-E0-E2),PCR和western blot检测表明,E0-E2基因已重组于腺病毒基因组中并获得表达。将rAd-E0-E2接种于小鼠和猪,并通过ELISA进行抗体检测。另外,将rAd-E0-E2经肌肉2次(间隔7d)免疫接种6周龄~7周龄猪,3周后用103TCID50CSFV石门株攻毒。结果表明,rAd-E0-E2免疫组6/7头存活,各组织器官带毒时间不超过10d,而非重组腺病毒rAd-CMV免疫组和空白对照组全部死亡,各组织器官均能分离到CSFV。结果提示,rAd-E0-E2能使免疫猪抵抗CSFV强毒攻击,为进一步研制猪瘟基因工程疫苗提供了实验依据。  相似文献   

6.
为制备猪瘟特异性抗体,将猪瘟兔化弱毒E2基因序列修饰后克隆到pFastBac1质粒载体,经转座、转染后构建重组杆状病毒,并用纯化的重组猪瘟E2蛋白免疫大耳白兔制备特异性抗体。实验结果表明成功获得能分泌重组猪瘟E2蛋白的杆状病毒,使用纯化的重组猪瘟E2蛋白制备的猪瘟抗体具有良好的特异性和敏感性,为猪瘟病毒荧光抗体染色液的制备奠定了基础。  相似文献   

7.
为了评价猪瘟E2亚单位疫苗单剂量免疫效果,本试验在试验猪场用猪瘟E2亚单位疫苗与猪瘟减毒活疫苗分别免疫供试猪,并定期测定猪瘟病毒抗体。饲养至24周龄时,每组随机选取5头运至检验动物房,用猪瘟石门系强毒进行人工感染。根据试验猪攻毒后临床症状、病理剖检和猪瘟抗原、抗体检测结果,比较分析了猪瘟E2亚单位疫苗单剂量免疫保护力。结果显示,对照组的试验猪攻毒后12 d内全部死亡,并表现出典型的猪瘟症状和病理变化;免疫猪全部存活,未见猪瘟症状和肉眼可见的组织器官病变,血样的猪瘟抗原ELISA检测结果均为阴性。结果表明,猪瘟E2亚单位疫苗单剂量免疫1次后,24周仍能为靶动物提供可靠的免疫保护效力,与猪瘟减毒活疫苗2次免疫的保护效力相当。  相似文献   

8.
在某个存在多种病毒性病原混合感染的规模猪场,通过使用猪瘟E2基因工程亚单位疫苗与猪瘟ST传代弱毒细胞苗进行免疫对比试验。免疫前该猪场猪瘟抗体水平比较低(平均ELISA阻断率低于40%),猪群表现为不稳定,存活率低,并且病原检测到猪蓝耳病病毒和猪圆环病毒2型;与弱毒苗相比较,猪瘟E2亚单位疫苗免疫后,猪瘟抗体滴度显著升高,猪群成活率明显提升。临床应用结果表明E2亚单位疫苗可以突破免疫抑制的影响,在猪群存在猪蓝耳病和猪圆环病毒病等免疫抑制性疾病的情况下,仍可以稳定提高猪瘟抗体水平,提供有效保护,提升生产成绩。  相似文献   

9.
为研究猪霍乱沙门菌C500(S.C500)运载猪瘟病毒(CSFV)新型基因疫苗口服免疫家兔的体内免疫应答特点,采用电转化法将CSFV新型基因疫苗转化到S.C500,构建重组工程菌,口服免疫接种家兔,检测CSFV、S.C500特异性抗体;并用猪瘟兔化弱毒疫苗及猪伤寒沙门菌野毒株依次进行攻毒试验。结果显示,成功构建CSFV新型基因疫苗重组菌S.C500/pCB-ME2-IL-15,S.C500/pCC-ME2-IL-15。口服免疫家兔可以诱导产生抗CSFV和S.C500的特异性ELISA抗体,且S.C500/pCC-ME2-IL-15略显优势。经三免后免疫家兔能够部分抵抗猪瘟兔化弱毒疫苗与猪伤寒沙门菌野毒株的攻击。试验提示以S.C500为CSFV新型基因疫苗运载体的猪用重组活菌苗具有可行性。  相似文献   

10.
本实验室前期构建了具有标记特征的腺病毒/甲病毒复制子嵌合载体猪瘟疫苗rAdV-SFV-E2,效力评价结果表明该嵌合疫苗的免疫原性与C株相当,并且能够完全提供对猪瘟病毒(CSFV)强毒石门株的攻毒保护。为进一步确定该疫苗的免疫效力是否受其母源抗体(MDA)的干扰及制定合理有效的免疫程序,本研究将rAdV-SFV-E2疫苗株免疫妊娠期的母猪后检测其抗体诱导水平,然后将该疫苗株免疫具有其母源抗体的30日龄新生仔猪及无母源抗体仔猪(n=4),攻毒后,监测所有仔猪的抗体水平、临床症状、病毒血症、病理学等。结果显示,有无母源抗体的仔猪均能够产生高水平的特异性E2抗体及抗CSFV的中和抗体,而且攻毒后未出现猪瘟特异性临床症状,包括发热、食欲减退、腹泻及死亡等;对照组仔猪均出现了明显的临床症状,其死亡率为100%。本研究证明rAdV-SFV-E2疫苗的免疫效力未受其母源抗体的显著干扰,免疫30日龄的仔猪后能够完全抵抗致死性CSFV强毒石门株的攻击。  相似文献   

11.
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which causes significant economic losses to the pig industry worldwide. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. This study was aimed to develop a recombinant human adenovirus type 5 expressing the CSFV E2 gene (rAdV-E2) and evaluate its efficacy in rabbits and pigs. The results showed that the rabbits and the pigs immunized with the rAdV-E2 developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits were protected from fever induced by infection with C-strain, which is pathogenic to the rabbit, and the rAdV-E2-immunized pigs were protected from lethal challenge with highly virulent Shimen strain. This indicates that the recombinant adenovirus can be an attractive candidate vaccine for preventing CSF.  相似文献   

12.
Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 105 TCID50 (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were Erns antibody negative and had seroconverted against Erns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.  相似文献   

13.
The aim of this study was to determine the immunomodulatory effects of IL-12, IL-18 and CD154 (CD40 ligand, CD40L) in DNA-vaccination against the classical swine fever virus. Four recombinant plasmids were constructed including the CSFV coding region for the glycoprotein gp55/E2 alone or together with porcine IL-12, IL-18 or CD154 genes. Five groups of four pigs each were immunized intramuscularly (i.m.) three times with the respective constructs. The control group was inoculated with empty plasmid DNA. Eighteen days after the final immunization, the pigs were challenged with a lethal dose of CSFV strain Eystrup and monitored for a further 16 days. This study showed that co-delivery of IL-18 and CD154 induced an earlier appearance of serum antibodies, reduced B-cell deficiency after infection and protected pigs against a lethal CSFV infection. In contrast, co-delivery of IL-12 led to a reduced titer of neutralizing antibodies and protection against a lethal CSFV challenge in comparison to the other pigs and to pigs that were immunized with a gp55/E2 plasmid alone.  相似文献   

14.
The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation, that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). Our results confirm the role of CCL20 to increase antibody-mediated responses. At the same time suggest the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs previously reported. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN. Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge. The levels of IFN-alpha detected in pigs immunized with pE2 and principally in non-vaccinated challenged animals can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed. Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV. To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the immune induction and protection against virus challenge in swine infection model.  相似文献   

15.
Li GX  Zhou YJ  Yu H  Li L  Wang YX  Tong W  Hou JW  Xu YZ  Zhu JP  Xu AT  Tong GZ 《Veterinary microbiology》2012,156(1-2):200-204
The amino acid sequence (TAVSPTTLR, 829-837aa) on the glycoprotein E2 of classical swine fever virus (CSFV) is a conserved and linear neutralizing epitope. In the present study, two peptides were constructed based the core sequence of this neutralizing epitope, the dendrimeric peptide (Th-B(4)) containing four copies of B cell epitope fused to one copy of promiscuous T helper (Th) cell epitope and the peptide Th-B containing a single copy of B cell epitope fused to one copy of Th cell epitope. The dendrimeric peptide Th-B(4) elicited high titers of neutralizing antibodies as detected in an indirect ELISA, blocking ELISA and neutralization test and induced a complete protection against CSFV C strain in rabbits. The Th-B elicited low titers of neutralizing antibodies and did not induce a protection in rabbits. These results suggest that the dendrimeric peptide Th-B(4) may be a promising marker vaccine candidate against CSFV and the multimerization is a requirement for development of a peptide vaccine.  相似文献   

16.
This study was designed to evaluate the prime-boost vaccination regimens as a novel immunization strategy for DNA vaccine against classical swine fever virus (CSFV). BALB/c mice were primed with the alphavirus replicon-vectored DNA vaccine pSFV1CS-E2-UL49 encoding the E2 protein of CSFV fused with the UL49 gene encoding the transduction protein VP22 of pseudorabies virus, followed by either homologous boosting with pSFV1CS-E2-UL49 or heterologous boosting with the recombinant adenovirus rAdV-E2 expressing the E2 protein or with the baculovirus-produced recombinant E2 protein (rE2) in adjuvant. The humoral and cell-mediated immune responses following prime-boost vaccination were assessed. The results showed that: (1) boosting with either rAdV-E2 or rE2 elicited high-level antibodies, whereas homologous boosting with pSFV1CS-E2-UL49 elicited low-level antibodies (below positive threshold); (2) heterologous boosting with rAdV-E2 resulted in stronger CD8+ and CD4+ T cells proliferation responses and higher stimulation indexes; and (3) heterologous boosting with rAdV-E2 induced more IFN-γ production. These results support the notion that a regimen of DNA prime-recombinant adenovirus boost enhances humoral and cell-mediated immune responses, and the DNA prime-protein boost regimen enhances humoral immune responses.  相似文献   

17.
In Thailand, where vaccination is routinely employed, there has been an increased incidence of chronic classical swine fever (CSF) outbreaks during the past decade. The major causative virus has been identified to be the moderate virulence, classical swine fever virus (CSFV) of the genogroup 2.2. An investigation was made into the efficacy of a CSF vaccine against this genogroup 2.2 challenge. Five-week-old pigs, grouped by their level of passive antibody titer were immunized with lapinized Chinese-strain CSF vaccine and challenged with CSFV genogroup 2.2, 13 days after vaccination. The group containing passive titers of lower than 64 at the time of immunization, had significantly higher number of CSFV-specific IFN-gamma secreting cells and was completely protected against the challenge. Interestingly, both cellular and antibody responses were inhibited in the pigs with the higher passive titer. Furthermore, following challenge, CSFV could be isolated from 50% of the pigs in this group. It was demonstrated that the CSF vaccine could induce complete protection in pigs, provided that the maternal derived titer at the time of vaccination was lower than 64. The result implied that an increase in CSFV outbreaks might be due to the inappropriate timing of vaccination as well as the nature of the CSFV genogroup 2.2.  相似文献   

18.
Weaned pigs (6-week-old) and 7-day-old pre-weaned piglets were vaccinated with naked plasmid DNA expressing the gp55/E2 gene from classical swine fever virus (CSFV). Both groups of pigs were then given a booster dose of recombinant porcine adenovirus expressing the gp55 gene (rPAV-gp55). Following challenge with CSFV, 100% of weaned pigs and 75% pre-weaned piglets were protected from disease. Weaned pigs given a single dose of rPAV-gp55 were also protected, but showed a slight increase in temperature immediately post-challenge. However, weaned animals given a DNA prime before rPAV-gp55 showed no fluctuation in body temperature following challenge and no pathology in spleen or lymph nodes upon post-mortem. In addition, no CSFV could be re-isolated from the rPAV vaccinated group and from only one pig in the prime-boost group following challenge, suggesting that both vaccination regimes have the potential to reduce or prevent virus shedding following experimental challenge.  相似文献   

19.
为制备抗猪瘟病毒(CSFV)单克隆抗体(MAb),本实验以表达CSFV E2囊膜糖蛋白的水泡口炎假病毒免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合;利用间接ELISA方法和携带荧光素酶(Luciferase)报告基因的HIV-luc/CSFV-E1E2假病毒系统筛选分泌中和性E2MAb的杂交瘤细胞;测定MAb亚型并纯化后,间接ELISA方法测定MAb的效价;采用western blot鉴定MAb的特异性亲和力;利用HIV-luc/CSFV-E1E2假病毒进行体外中和试验,分析MAb抑制病毒感染的能力。结果表明本实验获得了1株分泌中和性MAb的杂交瘤细胞9C8,该MAb能与E2蛋白特异性结合,而且体外抑制试验中和效价大于1∶25600。  相似文献   

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