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1.
Vaccination of chickens with DNA vaccine expressing Eimeria tenella MZ5-7 against coccidiosis 总被引：1，自引：0，他引：1
A chimeric DNA vaccine co-expressing Eimeria tenella MZ5-7 and chicken IL-17 gene was constructed and its efficacy against E. tenella challenge was evaluated. The ORF of MZ5-7 from E. tenella's second generation merozoite and the mature interleukin 17 gene of chicken were cloned into the expression vector of pcDNA4.0 to construct DNA vaccine pcDNA4.0-MZ and pcDNA4.0-MZ-IL17. The expression of aim gene products in vivo was detected by western blotting. The expression of IL-2 and IFN-γ in chicken splenocytes was detected by RT-PCR 7 days post-immunization. The expression levels of the two cytokines in the pcDNA4.0-MZ-IL17 DNA vaccine immunized group were significantly higher than that in the pcDNA4.0-MZ immunized group (p<0.05). Either pcDNA4.0-MZ or pcDNA4.0-MZ-IL17 DNA vaccine could obviously alleviate cecal lesions, body weight loss and increase oocyst decrease ratio. The ACI of pcDNA4.0-MZ-IL17 group was 190, which is higher than that of pcDNA4.0-MZ group and all the three control groups. In short, MZ5-7 was an effective candidate antigen for vaccine and co-expression of cytokine with antigen was an alternative method to enhance the immunity DNA vaccine. 相似文献
2.
In an attempt to investigate the immune efficacy ofa DNA prime-protein booster strategy against avian coccidiosis with a chimeric construct, the Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-gamma) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and prol, and then linked by splicing overlap extension by polymerase chain reaction to construct the chimeric plasmid prolE; the chimeric protein (rlE) was expressed in Escherichia coli harboring the constructed plasmid pGEX/IE. Broilers were administered two intramuscular injections with the constructed DNA vaccines (50 microg); in the protein booster groups 100 microg of the rlE were given following the proIE prime. After challenge the proIE-vaccinated chickens showed the protective immunity as demonstrated by significantly reduced oocyst shedding compared with chickens immunized with proE, but the prolE vaccine did not have an additive effect of increasing antibody titer and body weight gain. The chickens in the rlE booster groups had significantly higher specific antibody responses than those immunized with prolE, and displayed further decreased oocyst shedding and increased body weight gain. Taken together, these results indicate that ChIFN-gamma exerts an adjuvant effect coexpressed with 3-1E and provide the first evidence that the DNA prime-protein booster strategy is able to augment the protective efficacy of chimeric DNA vaccine against challenge with Eimeria tenella. 相似文献
3.
为深入研究乙型脑炎病毒（JEV）NS1和NS1-2A蛋白的表达和免疫效果差异,本试验构建并扩增C-端含Flag标签的NS1和NS1-2A基因,利用T4 DNA连接酶分别连接到质粒pcDNA3.1（+）上,构建重组质粒pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag。将这两种真核表达质粒分别转染BHK-21细胞,利用RT-PCR、IFA和Western blotting检测NS1和NS1-2A蛋白在体外的表达情况,用pcDNA3.1-NS1-Flag、pcDNA3.1-NS1-2A-Flag和pcDNA3.1（+）免疫BALB/c小鼠,检测这两种蛋白在体内的表达差异。结果显示,试验成功构建了NS1和NS1-2A基因的真核表达载体pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag,IFA和Western blotting鉴定NS1和NS1-2A蛋白成功表达,重组质粒pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag免疫小鼠后可诱发机体产生特异性体液免疫,pcDNA3.1-NS1-2A-Flag联合免疫组小鼠血清抗体效价和INF-γ细胞因子分泌水平比pcDNA3.1-NS1-Flag免疫组高,且与pcDNA3.1（+）空载体免疫组差异极显著（P<0.01）;pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag免疫组小鼠体内的INF-γ分泌量会增多,免疫第4周达到最高后逐渐降低。pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag免疫小鼠能够刺激JEV特异性抗体的分泌和增强机体的细胞免疫功能,且NS1-2A联合基因的免疫效果优于NS1单一基因,为进一步研究JEV的非结构蛋白功能、研发NS1和NS1-2A基因疫苗奠定基础。 相似文献
4.
为构建免疫调节型DNA疫苗,将山羊IL-2基因分别与捻转血矛线虫H11-1、H11-2和H11-3基因串联构建融合表达载体pcDNA/IL-2-H11-1、pcDNA/IL-2-H11-2和pcDNA/IL-2-H11-3。为检测疫苗在山羊体内的表达情况,将纯化的疫苗质粒肌肉注射山羊后取注射部位肌肉组织,于首次免疫7 d后和二次免疫10 d后分别用RT-PCR和Western Blot等方法检测H11抗原的转录和翻译情况。结果发现H11基因和IL-2基因均能在注射部位肌肉获得转录和翻译。 相似文献
5.
Construction of DNA vaccines encoding Eimeria acervulina cSZ-2 with chicken IL-2 and IFN-γ and their efficacy against poultry coccidiosis 总被引：1，自引：0，他引：1
Muhammad Ali A. Shah 《Research in veterinary science》2011,90(1):72-77
The study describes vaccination experiments with highly immunogenic sporozoite E. acervulina cSZ-2 co-administered with chicken IL-2 (chIL-2) and interferon-γ (chIFN-γ) to determine their efficacies against homologue challenge. The entire coding sequence of cSZ2, chIL-2 and chIFN-γ were cloned into eukaryotic expression vector pVAX1, constructing DNA vaccines pVAX1-cSZ2, pVAX1-chIL-2, pVAX1-chIFN-γ, pVAX1-cSZ2-chIL-2 and pVAX1-cSZ2-chIFN-γ. The expression of target genes in vivo was detected by RT-PCR and Western blot. Chicken experiments were carried out by vaccinating chickens two times at dose rate of 100 μg intramuscularly. At 28 days of age, all chickens were inoculated orally with 1 × 105 sporulated oocysts of E. acervulina except the unchallenged control group. Seven days after challenge, all chickens were weighted and slaughtered for duodenum collection. The results indicated that these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The findings also demonstrated best synergistic effect of IL-2 with this protein which suggested that co-administration of cytokines with this antigen was a powerful method to enhance immunity by alleviating intestinal lesions, body weight loss and oocyst count imparting partial protection against homologous challenge. 相似文献
6.
为研制水貂阿留申病核酸疫苗,应用重叠延伸PCR技术去除ADV VP2基因中编码428~446位氨基酸的核苷酸序列,与pc DNA3.1(~+)载体连接,构建全基因突变重组质粒pc DNA3.1-ADV-428,在此基础上,截去编码487~501位氨基酸的核苷酸序列,构建全基因突变重组质粒pc DNA3.1-ADV-428-487。将构建的重组质粒经肌肉注射免疫小鼠,应用间接ELISA法检测接种后14、28、42、56 d抗ADV抗体水平;流式细胞术检测接种后第42天小鼠脾细胞CD3~+、CD4~+和CD8~+T淋巴细胞亚群。结果显示,小鼠接种质粒后CD3~+、CD4~+和CD8~+T淋巴细胞亚群数量均明显增加,第42天抗ADV抗体水平达峰值。本试验通过对ADV全基因突变重组质粒的免疫原性进行分析,为水貂阿留申病核酸疫苗的研制提供了参考。 相似文献
7.
捻转血矛线虫Hc38基因DNA疫苗对绵羊免疫保护性效果评价 总被引：1，自引：0，他引：1
为了研究基因疫苗对绵羊的免疫保护效果,本研究构建了捻转血矛线虫(H.contortus)Hc38基因DNA疫苗.将H.contortus Hc38基因保守结构域克隆到真核表达载体pcDNA3.1中,免疫鼠8d后用RT-PCR检测到该疫苗在鼠肌肉组织中进行了转录.将纯化的DNA疫苗免疫绵羊后,用western blot和ELISA方法检测疫苗在绵羊体内的翻译和诱导IgG的产生.二免后2周用10 000条H.contortus第3期幼虫攻击实验动物,检测绵羊粪便虫卵排出、成虫数量等免疫保护性指标.该H.contortus Hc38 DNA疫苗与对照组比较,免疫组绵羊排出虫卵减少66.6%、成虫减少33.1%.特别值得注意的是免疫组的静脉注射方式产生抗体最高,相应羊的虫卵数和成虫数低.本实验证明Hc38基因DNA疫苗对绵羊虫卵及成虫发育具有明显的抑制作用. 相似文献
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9.
猪黑素皮质激素受体-4(melanocortin-4 receptor,MC4R)基因是影响猪生长肥育的主效基因之一,为了进一步研究MC4R基因的生物学功能,制备高成活率转基因猪,通过采用条件性诱导表达载体系统,PCR方法扩增了猪MC4R基因完整编码区,四环素诱导表达系统(Tet-on)的启动子调控区与转录调节元件。采用双酶切和连接等方法将上述元件连接到pcDNA3.1(+)上,构建了重组质粒pcDNA3.1(+)-rtTA-PTight-MC4R。经双酶切和测序鉴定,结果显示片段正向连接且片段全长测序正确。试验成功构建了四环素诱导的pcDNA3.1(+)-rtTA-PTight-MC4R真核表达载体, 并能在时间特异性方面调控MC4R基因的表达。 相似文献
10.
新城疫病毒F基因的真核表达及其免疫原性检测 总被引：3，自引：0，他引：3
为了探讨F基因在DNA免疫防制新城疫(ND)中的免疫原性,将NDV Z株F基因插入到真核表达载体pcDNA3.1/V5-H is-TOPO中,构建了真核表达质粒pcDNA NDV ZF。在脂质体作用下将pcDNA NDV ZF转染CEF细胞,用间接免疫荧光试验检测,在CEF细胞中可见有大量F蛋白表达。将重组质粒以100μg/只的剂量肌注免疫SPF雏鸡,经间接ELISA试验检测二免前后的血清,结果证明,pcDNA NDV ZF基因可在SPF鸡体内诱导相应抗体的产生,具有特定的免疫原性。 相似文献
11.
LONG Chong-chong DENG Wei-xi MAO Yi-zhi ZENG Gui-ying WU Bo-tao XIONG Chao-li XU Mao-wen XIA Peng WEN Ming 《中国畜牧兽医》2016,43(11):2886-2891
To explore the feasibility of entering clinical trials of the goat Chlamydophila abortus eukaryotic plasmids,Chlamydophila abortus OmpA gene was amplified by PCR and cloned into the eukaryotic expressing plasmid pcDNA3.1(+）to construction the recombinant vetor.After identification by PCR and restriction enzyme digestion,this vector was transfected into PK-15 cells and its expression were observed by fluorescent antibody test,the distribution of serum antibodies and plasmid were detected in mice after the immunization of the recombinant vector and molecular adjuvant which was single-stranded DNA of E.coli bacterial genome.The results showed that the recombinant plasmid pcDNA3.1-OmpA was successfully constructed after detecting by PCR,enzyme digestion and sequencing.The OmpA gene was effectively expressed in PK-15 cells.The anti-OmpA antibody levels of the pcDNA3.1-OmpA with molecular adjuvant group was significantly higher than that in pcDNA3.1-OmpA group and the inactivated vaccine group at 14 d after immunization（P<0.05).This levels showed an upward trend following numbers of immunization and times.The highest levels was at 35 d after immunization.Then the antibody titers were gradually decreased which still maintain a higher antibody levels than before.The pcDNA3.1-OmpA could be detected in the heart,liver,spleen,kidney,lung,brain,jejunum and leg muscle of mice on 21 d after immunization,and couldn't be detected in any organs at 49 d after immunization.The results above indicated that the the recombinant vetor based on OmpA gene of Chlamydophila abortus was successfully constructed in this experiment,after joining the molecular adjuvant could induce to a higher level of serum antibodies in mice. 相似文献
12.
为探讨山羊流产嗜衣原体重组真核质粒进入临床试验的可行性,本试验用PCR方法扩增出山羊流产嗜衣原体OmpA基因,克隆至真核表达载体pcDNA3.1（+）中,经PCR和双酶切鉴定后,将重组质粒pcDNA3.1-OmpA转染至PK-15细胞,观察目的基因表达情况,并将制备的大肠杆菌基因组单链DNA作为分子佐剂与重组质粒共同免疫小鼠,检测重组质粒在小鼠体内分布情况及血清抗体水平。结果表明,经PCR、双酶切和测序鉴定表明成功构建重组质粒pcDNA3.1-OmpA;转染PK-15细胞后,荧光抗体试验结果证实重组质粒pcDNA3.1-OmpA得到有效表达。首免后14 d,重组质粒加分子佐剂组的抗体效价显著高于重组质粒组和灭活疫苗组（P<0.05）;随着免疫次数增加和时间推移,免疫小鼠抗体水平均呈现上升趋势,至35 d时达到最高峰,此后抗体滴度逐渐下降,但仍维持较高水平。首免后21 d,在小鼠心脏、肝脏、脾脏、肾脏、肺脏、脑、空肠和腿肌中均可检测到质粒的分布,此后逐渐消失,49 d在所检组织中均未检测到重组质粒的存在。表明试验成功构建了基于OmpA基因的山羊流产嗜衣原体重组真核质粒,且加入分子佐剂后可诱导小鼠产生较高水平的血清抗体。 相似文献
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增强DNA疫苗免疫效果的研究进展 总被引：3，自引：1，他引：2
随着DNA重组技术的发展和应用,DNA疫苗、病毒和细菌活载体疫苗等基因工程疫苗的研制日益成为医学领域的一大研究热点。DNA疫苗在许多方面优于传统的灭活苗和减毒苗,但其免疫效果的稳定性和确实性方面尚存不足。影响DNA疫苗免疫效果的因素很多,国内外的许多研究者在这方面作了大量有意义的工作。目前,多数研究者主要通过目的基因的选择、促进外源基因在体内表达、改善疫苗导入方式以及辅以免疫刺激序列和免疫佐剂等方面来提高DNA疫苗的免疫效果。如果DNA疫苗在免疫效果方面能得到大幅度的提高,则它进入临床使用大有前途。 相似文献
14.
为探究绵羊肺炎支原体（Mycoplasma ovipneumoniae,Mo）pcDNA3.1-TBP30-Hsp70融合表达质粒对小鼠细胞免疫应答影响,本试验构建了绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒。用已构建的pMD19T-P30和pMD19-Hsp70质粒为模板,采用基因定点突变（SDM）原理设计引物,应用SOE-PCR扩增目的基因片段,并将其定向克隆至表达载体pcDNA3.1（+）,构建重组质粒pcDNA 3.1（+）-TBP30和融合重组质pcDNA3.1（+）-TBP30-Hsp70。使用pcDNA3.1-TBP30、pcDNA3.1-TBP30-Hsp70、pcDNA3.1（+）和Elution Buffer对小鼠进行免疫,应用ELISA试剂盒检测小鼠血清中细胞因子白细胞介素-2（IL-2）、IL-4、干扰素-γ（INF-γ）分泌水平。结果显示,pcDNA3.1-TBP30-Hsp70酶切后可见大小分别约为1 413 bp的目的基因片段和5 400 bp的载体条带。与空白对照组和pcDNA3.1（+）组相比,免疫重组质粒组均可引起小鼠血清中细胞因子INF-γ、IL-2和IL-4分泌水平的增强,与空白对照组和pcDNA3.1（+）组相比差异显著或极显著（P<0.05;P<0.01）;而空白对照组和空质粒组之间差异不显著（P>0.05）;免疫pcDNA3.1-TBP30和pcDNA3.1-TBP30-Hsp70组小鼠血清IL-2、INF-γ和IL-4终分泌量增加,表明重组质粒组可刺激小鼠血清中IL-2、INF-γ和IL-4的变化,并且在时间上都呈现出先增多后减少的规律。本试验结果表明,重组质粒pcDNA3.1（+）-TBP30-Hsp70免疫小鼠后,IL-2和INF-γ分泌水平的升高,增强了机体的细胞免疫功能,进而调节机体细胞免疫影响T细胞和巨噬细胞的分泌,从而提高机体细胞免疫能力;IL-4分泌水平升高,促进机体Th2向Th1分化,维持Th1的优势状态,增强了机体的细胞免疫功能。本试验结果为绵羊肺炎支原体基因工程疫苗的研制提供了参考依据。 相似文献
15.
柔嫩艾美耳球虫马杜拉霉素抗药株与敏感株的mRNA差异显示 总被引：3，自引：0，他引：3
利用柔嫩艾美耳球虫马杜霉素敏感虫株裂殖子和抗药虫株裂殖子为材料,对马杜霉素敏感虫株和抗药虫株进行差异显示PCR,共回收34条电泳差异片段,反向Northern点杂交鉴定4个片段为真正差异片段,并对4个片段进行测序、B1ast同源性比较.来自于马杜霉素抗药虫株裂殖子的ACD3-2序列与柔嫩艾美耳球虫微线-5同源性99%,说明AcD3-2序列是该基因的部分序列,微线-5蛋白与虫体融解宿主细胞膜、入侵、运动和溢出有关,在第二代裂殖子时期,抗药性虫株该序列发生转录,而在敏感虫株中沉默;HCD1序列来自马杜霉素敏感虫株,与柔嫩艾美耳球虫表面抗原16和17序列同源性分别为83%和86%,说明与这2个基因同源.AGD5片段来自抗药虫株,HAD8-2序列来自敏感虫株,通过比较这2条序列可能为新序列. 相似文献
16.
鸡IL-2基因的克隆及GST-chIL2融合蛋白的表达 总被引：1，自引：1，他引：0
根据Sundick等发表的鸡IL-2基因(chIL2)序列设计合成特异性引物,用RT-PCR从ConA诱导的鸡脾淋巴细胞扩增出450 bp的目的片段,酶切鉴定及序列测定结果表明为鸡IL-2基因。该基因包括鸡IL-2基因的全部开放阅读框,编码142个氨基酸组成的蛋白质,与GenBank鸡IL-2基因相比,在编码氨基酸的49位有一个氨基酸缺失;而与Broiler、SC、Chenren和Xiaoshan鸡在编码氨基酸上完全一致,具有较近的亲缘关系;与Kestrel来航鸡、来航SPF鸡、Obese、Silky和Xianju鸡等有1-4个氨基酸的差异;与火鸡和鹌鹑的氨基酸同源性分别为69.9%和59.4%。将克隆到的基因插入到融合蛋白原核表达载体pGEX-6p-1中,得到重组表达质粒pGEX-IL2。将此重组质粒转化大肠杆菌DH5α,经IPTG诱导,表达出了大小约为40 ku的GST-chIL2融合蛋白,其中GST部分为26 ku,鸡IL-2为14 ku,与预期的鸡IL-2成熟蛋白大小一致。 相似文献
17.
CpG寡核苷酸对IBDV VP2基因真核表达质粒免疫增效作用 总被引：1，自引：1，他引：1
以传染性法氏囊病病毒(IBDV)VP2蛋白基因表达质粒DNA为免疫原，以CpG的寡核苷酸(CpG-0DN)为免疫佐剂，肌肉注射于14日龄SPF鸡，1周后加强免疫1次，2次免疫后15d和21d分别测定血清ELISA抗体效价，并于免疫后21d用IBDV99儿强毒株攻毒和进行病理学观察。结果显示，(1)VP2基因重组质粒DNA与CpG共同免疫组的ELISA抗体水平明显高于VP2重组质粒免疫组；(2)IBD弱毒苗与VP2重组质粒免疫组抗体水平明显高于VP2重组质粒免疫组，且比VP2基因重组质粒DNA与CpG共同免疫组略高；(3)VP2基因重组质粒DNA与CpG共同免疫组及IBD弱毒苗与VP2重组质粒免疫组可明显降低IBDV强毒攻击后引起的急性发病率和死亡率。由此表明，CpG寡核苷酸对IBDV VP2蛋白基因真核表达质粒免疫具有明显增强作用，有很大的应用前景。 相似文献
18.
Jazayeri SD Ideris A Zakaria Z Yeap SK Omar AR 《Comparative immunology, microbiology and infectious diseases》2012,35(5):417-427
This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study. 相似文献
19.
为获得紫茎泽兰处理鸡柔嫩艾美耳球虫后差异基因,将0.5%紫茎泽兰提取液作用于鸡柔嫩艾美耳球虫卵囊孢子化过程,采用抑制消减杂交技术（SSH）筛选处理后卵囊的差异表达基因,通过GO和COG功能预测分析,并采用实时荧光定量PCR验证差异表达的基因。结果显示,采用SSH成功构建了紫茎泽兰处理前后鸡柔嫩艾美耳球虫卵囊的cDNA消减文库,获得86条ESTs序列,经拼接和聚类后得到31条独立基因（Unigenes）,其中有23个基因有功能注释,8个基因没有功能注释,另外有4个基因没有同源性匹配。为进一步验证文库的特异性,从中随机选取3个差异表达基因,运用实时荧光定量PCR技术验证表面抗原13、3-羟酰辅酶A脱氢酶和细胞色素P450基因在紫茎泽兰处理前后的表达差异,结果显示,3个基因在经紫茎泽兰处理的鸡球虫孢子化卵囊中的表达量明显低于未处理组,说明紫茎泽兰对柔嫩艾美耳球虫卵囊具有一定的活性抑制作用。本试验获取了紫茎泽兰作用柔嫩艾美耳球虫卵囊的主要调控基因,为将紫茎泽兰研发成环境杀虫剂奠定了良好的理论基础,也可为球虫致弱疫苗或基因缺失疫苗的靶标筛选研究提供参考。 相似文献
20.
通过构建蓝舌病毒(BTV)NS4基因真核表达载体pcDNA3.1-NS4-eGFP,转染HEK-293T细胞,利用Western blot及荧光显微镜分析NS4蛋白的表达与亚细胞定位特征;pcDNA3.1-NS4-eGFP转染的HEK-293T细胞添加20 HAU/mL仙台病毒(SeV)刺激后,qRT-PCR法分析NS4基因表达对SeV诱导的上游识别基因RIG-Ⅰ、MDA5、VISA、TBK1、IKKε、IRF3、TRAF3、TRAF6、IRF9、干扰素基因(IFN-α、IFN-β)以及干扰素刺激基因ISG15和USP18的mRNA表达水平的影响。在HEK-293T细胞内转染pcDNA3.1-NS4-eGFP质粒24 h后,分别添加20 HAU/mL SeV刺激24,48 h,qRT-PCR结果表明,细胞内表达NS4-EGFP后,RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15及IFN-β基因mRNA表达极显著下降,随着SeV诱导时间的延长,VISA、TBK1、IKKε、USP18基因mRNA表达差异呈不显著趋势。本研究成功构建BTV NS4基因真核表达载体pcDNA3.1-NS4-eGFP,NS4-EGFP融合蛋白在HEK-293T细胞中主要分布于细胞核周围及细胞核内。BTV NS4基因在HEK-293T细胞内的表达显著下调SeV诱导的IFN信号通路相关基因RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15和IFN-β的表达,为进一步探究NS4基因在BTV拮抗宿主细胞免疫应答中的机制奠定基础。 相似文献