长链CpG寡脱氧核苷酸DNA重组质粒对禽流感灭活疫苗免疫应答的影响

为明确导入长链CpG寡脱氧核苷酸3个DNA重组质粒对禽流感H5亚型灭活疫苗的免疫应答的影响,将21日龄试验雏鸡随机分为6组,即健康对照组(A组)、疫苗对照组(B组)、空载体对照组(C组)、pUC26免疫促进组(D组)、pUC30免疫促进组(E组)及pUC44免疫促进组(F组),对6组雏鸡在首免后1～8周(WPI)检测其HI抗体水平并在1、4及8 WPI测定脾脏、法氏囊及胸腺免疫器官指数.研究结果表明:在2WPI,D(P<0.01)、F(P<0.01)及E组(P<0.05)HI抗体水平极显著或显著高于C组;在3WPI,F组HI抗体水平分别显著高于B(P<0.05)、C(P<0.05)及E组(P<0.05);在5WPI,F(P<0.01)和D(P<0.05)组HI抗体水平极显著或显著高于C组;在6 WPI,F组HI抗体水平显著高于C组(P<0.05);在8 WPI,F组HI抗体水平极显著高于B(P<0.01)或C组(P<0.01).在4WPI,A组胸腺指数极显著或显著高于C(P<0.05)及D组(P<0.01),E组胸腺指数显著高于D组(P<0.05);在8WPI,E组脾脏指数显著高于A组(P<0.05).     

CpG ODN重组质粒对猪外周血淋巴细胞免疫刺激作用的研究

为获得对猪具有免疫刺激活性的含CpG序列的重组质粒,设计合成了11条CpG序列,应用MTS比色法测定合成CpG ODNs刺激猪PBMC增殖的能力,结果有10条CpG ODN对猪PBMC具有刺激活性(SI>2);以对猪PBMC具有较高刺激活性的CpG06和CpG08为核心,分别合成含6次重复序列的重组质粒pCpG06和pCpG08,并检测其对猪PBMC的刺激活性,结果重组质粒pCpG06和pCpG08对猪PBMC的刺激指数分别可达4.97和5.32,并可刺激猪PBMC产生较高水平的IL-4。研究获得的重组质粒pCpG06和pCpG08对猪PBMC具有较好的刺激活性,可为猪用CpG佐剂的研究提供参考。     

CpG寡脱氧核苷酸的免疫刺激特性及应用研究进展

CpG基序是指含有非甲基化的胞嘧啶（C）和鸟嘌呤（G）二核苷酸为核心序列的基序为5′-R-D-CpG-Y-Y-3′的DNA或寡脱氧核苷酸序列,是DNA具有免疫刺激活性的结构基础。随着对CpG特性研究的不断深入,CpG寡脱氧核苷酸在激活先天性免疫、作为疫苗佐剂、对变态反应进行免疫治疗、抗肿瘤应用及基因治疗等方面均显示出较广阔的应用前景。现就CpG寡脱氧核苷酸的免疫激活作用、细胞识别及结构特征、治疗疾病方面的应用及毒理作用加以探讨。     

疫苗佐剂胞嘧啶-鸟嘌呤寡脱氧核苷酸的研究进展

胞嘧啶-鸟嘌呤寡脱氧核苷酸(cytosine phosphate guanidine oligodeoxynucleotide,CpG ODN)是指含有非甲基化的胞嘧啶和鸟嘌呤二核苷酸为核心序列的核苷酸序列,近年来,CpG ODN作为一种新型免疫佐剂的研究越来越多,可诱发机体产生多种免疫学效应,提高系统免疫和黏膜免疫水平,具有安全性高,耐受性强等特点。     

CpG ODN联合重组乳酸乳球菌鼻内免疫BALB/c小鼠的佐剂效应

本试验初次建立了CpG ODN联合重组乳酸乳球菌滴鼻免疫BALB/c小鼠模型。结果得出CpG ODN佐剂协同重组菌抗原组血清中抗PRRSV特异性抗体IgG和呼吸道黏膜抗体s-Ig A均高于单独重组菌组(P>0.05)和单独佐剂组(P<0.01),佐剂CpG ODN协同重组菌抗原组的Th1型细胞因子IL-2、IFN-γ及黏膜淋巴因子IL-5的活性高于单独抗原组(P>0.05)及单独佐剂组(P<0.01)。试验结果说明CpG ODN作为佐剂与重组菌鼻腔免疫小鼠后,可以提高重组菌诱导的黏膜免疫反应和Th1细胞参与的系统免疫反应,为研究安全有效的重组菌黏膜疫苗奠定了基础。     

线粒体DNA缺失质粒pMDD-Z的构建及鉴定

分别以pEGFP-Mito和pAN4质粒为模板,扩增出MTS-EGFP和EcoRⅠ基因片段,利用快速体外基因重组技术-重组PCR,将两者连接得到MTS-EGFP-EcoRⅠ片段,构建克隆载体pTRE2hyg-EE,经AgeⅠ和NotⅠ双酶切得到MTS-EGFP-EcoRⅠ融合基因产物,连接克隆于真核表达质粒pEGFP-Mito后获得pMDD-Z质粒。琼脂糖凝胶电泳结果显示,MTSEGFP和EcoRⅠ基因片段大小分别是831 bp和860 bp。测序结果显示,插入pTRE2hyg--EE克隆载体中的MTS-EGFPEcoRⅠ片段大小1 672 bp,且基因序列正确,没发生点突变。琼脂糖凝胶电泳结果显示,pMDD-Z质粒的双酶切产物分别为1 587 bp的插入片段和4 024 bp的载体片段。测序结果显示,pMDD-Z质粒中含1 659 bp的开放阅读框,编码553个氨基酸,从N端至C端,分别为线粒体信号导肽、绿色荧光蛋白和限制性内切酶EcoRⅠ,且未发生移码突变。     

一种克隆载体重组质粒pMD18-T Simple-PCV2的构建

为了研究猪圆环病毒2型(PCV2)的全基因组变异特性,试验设计了1对特异性引物,并从疑似断乳仔猪多系统衰竭综合征(PMWS)的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后形成重组质粒pMD18-T Simple-PCV2(命名为P-S-PCV2),经酶切鉴定、PCR鉴定和序列测定分析的阳性重组质粒被证明含有长为1 767 bp的PCV2片段,同时通过Blast与GenBank中国内外的部分PCV2毒株进行同源性比较分析。结果表明:与国内外12个毒株间的同源性介于95.0%～100%之间,与国内的河北株(HB株)亲缘关系最近,同源性高达100%;与澳大利亚株(AUT2株)亲缘关系最远,同源性为95.0%。说明来源不同国家和地区的猪圆环病毒2型毒株存在地域性差异。     

地高辛标记PPV—DNA—C及PUP重组质粒探针的比较研究

应用分子克隆技术制备的猪细小病毒复制型ＤＮＡ的ＰｓｔⅠ／ＨｉｎｄⅢ双酶切片段Ｃ及被克隆到ＰＵＣ１９中的Ｃ片段形成的重组质粒ＰＵＰ利用非放射性的地高辛标记后制备两种探针。分别对不同来源的猪细小病毒ＤＮＡ及ＰＵＰ重组质粒于硝酸纤维素膜上打点杂交，免疫呈色后均为阳性反应，而对照的猪瘟病毒、乙型脑炎病毒、伪狂犬病毒、ＰＫ－１５细胞的核酸均为阴性反应。通过对已知量的ＰＰＶ－ＤＮＡ检测发现Ｃ探针及ＰＵＰ探针的最低检出限量分别为４０ｐｇＤＮＡ和４ｐｇＤＮＡ。重组质粒ＰＵＰ探针比双酶切后的Ｃ片段探针敏感１０倍     

鸡痘病毒282E_4株TK基因重组载体质粒的构建

应用已克隆的鸡痘病毒（ＦＰＶ）２８２Ｅ＿４株基因组ＴＫ基因，在ＮｃｏⅠ部位引入痘苗病毒Ｐ７．５启动子和Ｐ１１启动子控制下的大肠杆菌ＬａｃＺ基因，经酶切鉴定，成功地构建了用于ＦＰＶ重组的载体质粒。与亲本毒株在鸡胚成纤维细胞内共转染，产生了表达ＬａｃＺ基因的重组鸡痘病毒。经传代筛选、纯化，重组病毒较稳定。实验结果表明，以ＴＫ基因构建的重组载体质粒可用于外源基因的重组表达研究。     

免疫佐剂 CpG—DNA的研究进展

特异性免疫即是含有PAMP的反应物与天然免疫细胞上的PRR相结合,激活天然免疫应答,从而活化抗感染途径,并最终发挥获得性免疫应答的一系列反应过程。CpG—DNA作为一种在进化中高度保守的PAMP,它可以模拟感染过程,克服新型疫苗PAMP不足的缺陷,从而诱导强烈的免疫应答。CpG—DNA可以直接激活B淋巴细胞、单核／巨噬细胞和树突状细胞,上调免疫刺激分子的表达,调节免疫反应,诱导IL-12、IL-1和TNF-等多种细胞因子的分泌,此外,它还能间接刺激NK细胞、T淋巴细胞。由于其独特的优点,CpG—DNA在畜牧兽医临床中显示其极强的使用价值和广阔的应用前景。     

鸡CD3γδ基因cDNA的克隆及其真核表达质粒的构建

用Trizol提取4—6周龄白莱航鸡、伊莎鸡、固始鸡和隐性白羽鸡胸腺组织的总RNA，再用Oligo～dT纤维素富集mRNA后，用RT-PCR扩增出鸡CD3γδ基因CDNA。PCIk产物切胶回收后连入T载体，序列分析发现伊莎鸡、固始鸡和隐性白羽鸡的CD318cDNA比白菜航鸡多一个氨基酸，且在胞外区的一个区域有4个氨基酸的差异。将白莱航鸡的CD318从T载体上切下连入pcDNA3．1（＋），再将重组质粒pcDNA3．1-CD3转染COS-7细胞，用已知的抗鸡CD3的单克隆抗体测得转染细胞中CD3γδ分子的表达，这说明构建的真核表达质粒正确，为下一步用DNA免疫的手段研制抗鸡CD3的单克隆抗体以及研究鸡CD3分子的结构和功能打下了基础。     

CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferation.

Oligodeoxynucleotides (ODNs) with unmethylated CpG dinucleotide motifs may be useful as non-specific immune system stimulants and adjuvants for protein or nucleic acid vaccines in humans and other primates. They may also be useful in cancer immunotherapy and in the modulation of allergic responses or mucosal immunity. To begin to determine the potential utility of CpG ODN technology in small animal veterinary medicine, we developed procedures to analyze the effects of CpG ODN on canine and feline blood, spleen and lymph node (LN) cells. We find that certain CpG ODN cause good lymphocyte proliferation (as monitored by [(3)H]-thymidine incorporation) in both canine and feline spleen and LN cells, but not in blood. This overall stimulatory effect of CpG ODN on spleen and LN cells is CpG dependent. The reverse sequences, GpC ODNs, do not cause significant lymphocyte proliferation in the cat; however, dogs are more sensitive to stimulation by the non-specific immune effects of the phosphorothioate backbone. We conclude that unmethylated CpG ODNs may also have potential uses as immune stimulants for vaccines and other antimicrobial agents in veterinary medicine for companion animals.     

Protection of chickens against a lethal challenge of Escherichia coli by a vaccine containing CpG oligodeoxynucleotides as an adjuvant

Synthetic oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs (CpG-ODN) have been shown to be effective immunoprotective agents and vaccine adjuvants in a variety of bacterial, viral, and protozoan diseases in different animal species. The objective of this study was to compare the immune response of chickens to a killed Escherichia coli vaccine combined with oil in water emulsion or with CpG-ODN. Birds were vaccinated with killed E. coli antigens with either 10 or 50 microg of CpG-ODN on days 10 and 20 of age. At day 30, a virulent isolate of homologous E. coli was applied on a scratch site on the caudal abdominal region. Birds were examined for 10 days post-E. coli challenge, and pathologic and bacteriologic assessments were conducted on all birds that were either found dead or euthanized. The E. coli vaccine group that received no CpG-ODN had a survival rate of 65%. In contrast, groups that received the vaccine with CpG-ODN adjuvant had significantly higher survival rate of 92% (P < 0.01) with isolation of low numbers of E. coli from internal organs. Total IgG against E. coli antigens was highest in groups that received CpG-ODN as an adjuvant. Birds that received vaccine containing CpG-ODN had minimal inflammatory reaction without tissue necrosis at the injection site. Severe tissue necrosis was present in birds that received vaccine containing oil in water emulsion adjuvant. This study demonstrated that CpG-ODN is an effective vaccine adjuvant in chickens and results in minimal tissue destruction. This study is the first study in which CpG-ODN has been demonstrated to produce an adaptive immune response, at a significant level, against an extracellular bacterial infection in chickens.     

ADV分离强毒株全基因突变重组质粒的构建、表达及其免疫原性的分析

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。     

猪繁殖与呼吸综合征DNA重组质粒对猪的免疫效应

猪繁殖与呼吸综合征病毒(PRRSV)灭活疫苗、表达GP5蛋白的DNA重组质粒分别与表达IL-2和IL-4的重组质粒(pcDNA-IL-2和pcDNA-IL-4)联合免疫健康仔猪,经3次免疫后人工感染PRRSV HB-2株,检测仔猪体液免疫以及攻毒保护性反应。研究结果显示,重组质粒pCI-GP5可诱导免疫猪产生抗GP5抗体,最高ELISA抗体效价可达1∶285。攻毒后组织中PRRSV核酸的检出率下降30.3%,与对照组相比,差异显著(P<0.05),表明表达PRRSV GP5的DNA重组质粒pCI-GP5可诱导一定的免疫效力。pcDNA-IL-2与pCI-GP5联合免疫后,病毒血症的出现频率减少38.9%,PRRSV阳性组织检出率下降28.8%,与对照组差异显著(P<0.05);pcDNA-IL-4与pCI-GP5联合免疫后,最高ELISA抗体效价可达1∶320,病毒血症的出现频率下降38.9%,PRRSV阳性组织检出率减少34.8%,与对照组相比差异极显著(P<0.01)。本研究表明,PRRS DNA重组质粒pCI-GP5对猪的免疫保护力是稳定的,真核表达的细胞因子pcDNA-IL-2与pcDNA-IL-4能够显著增强pCI-GP5的免疫保护力。     

Protection of neonatal broiler chicks against Salmonella Typhimurium septicemia by DNA containing CpG motifs

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG-ODN) motifs have been shown to stimulate the innate immune system against a variety of bacterial and protozoan infections in a variety of vertebrate species. The objective of this study was to investigate the immunostimulatory effect of CpG-ODN in neonatal broilers against Salmonella Typhimurium septicemia. Day-old broiler chicks, or embryonated eggs that had been incubated for 18 days, received 50 microg of CpG-ODN, 50 microg of non-CpG-ODN, or saline. Four days after exposure to CpG-ODN or day 2 posthatch, 1 x 10(6) or 1 x 10(7) colony-forming units (cfu) of a virulent isolate of Salmonella Typhimurium was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 10 days following challenge with Salmonella Typhimurium. The survival rate of birds in groups receiving either non-CpG-ODN or saline following Salmonella Typhimurium infection was 40%-45%. In contrast, birds receiving CpG-ODN had significantly higher survival rate of 80%-85% (P < 0.0001). Bacterial loads and pathology were low in groups treated with CpG-ODN compared to the groups receiving saline or non-CpG-ODN. Colony-forming units of Salmonella Typhimurium in the peripheral blood were significantly lower in birds treated with CpG-ODN compared to the group that received saline. This is the first time that CpG-ODN has been demonstrated to have an immunoprotective effect against an intracellular bacterial infection in neonatal broiler chickens following in ovo delivery.     

Systemic innate immune responses following intrapulmonary delivery of CpG oligodeoxynucleotides in sheep

Mucosal delivery of CpG oligodeoxynucleotide (ODN) in mice has been shown to induce potent innate immunostimulatory responses and protection against infection. We evaluated the efficacy of CpG ODN in stimulating systemic innate immune responses in sheep following delivery to the pulmonary mucosa. Intrapulmonary (IPM) administration of B-Class CpG ODN in saline induced transient systemic responses which included increased rectal temperatures, elevated serum 2'5'-A synthetase and haptoglobin concentrations. The ODN dose required to induce detectable systemic responses following IPM delivery could be reduced by approximately 80% if the CpG ODN was administered in 30% emulsigen instead of saline. Intrapulmonary B-Class CpG ODN formulated in 30% emulsigen produced similar effects when compared to those seen following SC injection. These responses were CpG ODN-specific since control GpC ODN did not induce any detectable response. Intrapulmonary administration of both B-Class and the newly described C-Class CpG ODN produced similar effects indicating that both classes of CpG ODN were comparably effective in stimulating innate immune system following mucosal delivery. Administration of CpG ODN directly into the lungs or delivery of CpG ODN via an intratracheal (IT) infusion also produced similar systemic responses. These observations support the conclusion that mucosal delivery of CpG ODN is an effective route for induction of systemic acute phase responses and antiviral effector molecules in large animals, and may be helpful in controlling systemic infections.     

FMDV全ORF编码基因的克隆及其腺病毒穿梭质粒的构建

为研制FMD复制缺陷型腺病毒活栽体疫苗，通过RT—PCR方法获得了。型口蹄疫病毒（FMDV）全开放阅读框（oRF）基因片段，将其克隆到pMD18-T栽体后测序，再将oRF编码基因定向克隆入腺病毒穿梭载体pAdTrack—CMV中，构建了重组腺病毒穿梭质粒。经PCR、酶切及测序鉴定，所克隆的oRF编码基因序列与原始强毒株Akesu／58的ORF编码基因比较，L、P1、P2、P3基因的核苷酸同源性分别为98．8％、97．9％、99．0％和97．6％，PCR、酶切鉴定重组腺病毒穿梭质粒均获得了长约6．9kb的目的基因片段。表明，成功获得了含有。型FMDV全ORF编码基因的阳性克隆，并成功构建了含有完整FMDV开放阅读框架的编码基因表达盒的重组腺病毒穿梭质粒。     

奶牛乳腺炎源性大肠杆菌csgC基因的克隆及载体构建

根据GenBank上发表的curli菌毛csgC基因序列设计了1对特异性引物.从患乳腺炎的奶牛乳汁中分离出致病性大肠杆菌,经生物学鉴定后,提取全基因组DNA为模板,PCR扩增出csgC基因,连入pMD18-T克隆载体,测序,结果表明,扩增片段含有333个核苷酸,编码111个氨基酸的成熟蛋白,与已报道的大肠杆菌W3110的全基因组DNA中的csgC基因序列最相近,氨基酸序列同源性为99.7%.该基因与原核表达载体pET32a+相连,转入BL21(DE3)感受态细胞.挑选阳性菌落经PCR鉴定和酶切鉴定后表明成功构建原核表达载体pET32a+/csgC.     

Immunoadjuvant effects of bacterial genomic DNA and CpG oligodeoxynucleotides on avian influenza virus subtype H5N1 inactivated oil emulsion vaccine in chicken

This study investigated the immunoadjuvant effects of three types of bacterial genomic DNA and CpG oligonucleotides (CpG ODN) on the avian influenza virus (AIV) subtype H5N1 inactivated oil emulsion vaccine under two immunization strategies. The genomic DNA extracted from Escherichia coli O₂, Staphylococcus aureus, Streptococcus faecalis FQ68, and synthetic CpG ODN were used as adjuvants, and their effects on the AIV oil emulsion vaccine were examined in chickens. The results indicated that when administered separately from the vaccine, adjuvants induced lower haemagglutination inhibition (HI) titres and serum IgG titres but resulted in higher concentrations of IFN-γ and IL-10. In contrast, when combined with the oil emulsion vaccine prior to inoculation, CpG ODN induced higher HI, IgG titres and IFN-γ concentration but resulted in lower IL-10 concentration. These data suggest that, depending on the immunization approaches, adjuvants may exert distinct immune effects in chickens receiving AIV H5N1 oil emulsion vaccine: the prior incorporation of CpG ODN into the vaccine may augment both the humoral and Th1 type immune responses, while separate inoculation of adjuvants has not shown better adjuvanticity.