山地乌骨鸡IL-1β基因克隆测序及对IBV DNA疫苗免疫佐剂效应研究
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摘要
本研究根据Genebank中鸡白介素-1β(ChIL-1β)序列(Y15006)设计引物对，采用RT-PCR法从地方品种山地乌骨鸡的外周血淋巴细胞RNA中克隆了ChIL-1β基因，将cDNA片段克隆到PMD18-T载体，进行序列测定，并对其进行分析。山地乌骨鸡IL-1β序列在国内外是第四个报道的鸡IL-1β序列。分析结果显示：该克隆的cDNA全长804bp，编码267个氨基酸。将山地乌骨鸡IL-1β基因测序结果与Genebank上的仅有的1株IL-1β序列(Weining，K.C.等报道的序列)用DNAstar进行分析发现序列同源性99.5％，与国内邵卫星等报道的IL-1β序列同源性为99.0％，与张永霞等报道的IL-1β基因序列同源性为97.9％，将其编码的氨基酸进行同源性分析，同时将4个核苷酸序列进行进化树分析。该基因经BamHⅠ和E_(CO)RⅠ双酶切后，插入同样经BamHⅠ和E_(CO)RⅠ双酶切真核表达载体pcDNA3.1(+)中，构建真核表达质粒，经鉴定正确，配制分子佐剂pDNAIL-1β，与IBV DNA疫苗联合免疫7组鸡(pcDNA3.1组，IBV DNA组，pDNAIL-1β组，pDNAIL-1β+IBV DNA组，PBS组，灭活苗组，弱毒苗组)。
用间接ELISA测定特异性抗体，结果表明，免疫14天时pDNAIL-1β+DNA组和弱毒苗组、灭活苗组比较差异极显著(P＜0.01)，在21天pDNAIL-1β+IBV DNA组与所有实验组比较差异极显著(P＜0.01)，在28天pDNAIL-1β+DNA组与DNA疫苗组比较差异显著(P＜0.05)；测定CD3+T淋巴细胞结果表明，pDNAIL-1β+IBV DNA组与IBV DNA疫苗组比较在14天差异极显著(P＜0.01)，21天时pDNAIL-1β+IBV DNA组与IBV DNA疫苗组比较差异显著(P＜0.05)，pDNAIL-1β+IBV DNA组与其它实验组比较均出现差异极显著(P＜0.01)；检测CD4+T淋巴细胞结果表明，7天时pDNAIL-1β+IBV DNA组与pcDNA3.1组、灭活苗组、弱毒苗组和PBS苗组比较差异显著(P＜0.05)，在14-21天期间pDNAIL-1β+IBV DNA组与IBV DNA疫苗组比较，差异极显著(P＜0.01)；研究CD8+结果表明，在免疫14天，pDNAIL-1β+IBV DNA组和IBV DNA疫苗组比较差异极显著(P＜0.01)，21天时与除DNA疫苗组外的其它五个实验组比较差异显著(P＜0.05)。
攻毒实验结果表明，pDNAIL-1β+IBV DNA组其死亡保护率(95％)显著高于IBV DNA疫苗组(85％)，与弱毒苗组的保护率相当(95％)，但低于灭活苗组的保护率(100％)。
综上表明pDNAIL-1β可以明显地增强IBV DNA疫苗的免疫作用，可作为一种有效的免疫增强剂。
We designed the primers based on chicken interleukin-1βin Genebank(Y15006),and then Gallus domesticus Brisson interleukin-1βwas amplifiedby recerse transcription-polymerase chain reaction(RT-PCR)from peripheralblood lymphocytes stimulated with ConA. The gene was cloned into PMD18-T vectorand then sequenced. Gallus domesticus Brisson IL-1βwas the fouth nucleotidesequence reported in the world. The result showed that the length of cDNA was804bp, encoding 267 amino acids, sharing 99.5% homology with the ChIL-1βgeneof Weining, K. C. in Genbank. The nucleotide sequence shared 99.0% and 97.9%homology with the ChIL-1βgene of Shao Weixing and Zhang Yongxia clonedrespectively. Wecarried on the homology analysis to those encoded amino acidand the evolution tree analysis 4 nucleotides sequences. Then digested thegene with BamHⅠand E_(CO)RⅠ, inserted into pcDNA3.1(+) which was also digestedwith BamHⅠand E_(CO)RⅠ. The eukaryatic expression vector of chickeninterleukin-1βgene (pDNAIL-1β) was constructed. By identified accuracypDNAIL-1βcould be used as an immune intensifier to immunity 7 groups ofchickens(pcDNA3. Igroup, IBV DNA group, pDNAIL-1βgroup, pDNAIL-1βplus IBVDNA group, PBS group, inactive IBV vaccine group, attenuated group).
With indirect ELISA admeasurement, peculiarity antibody of pDNAIL-1βplusIBV DNA group and inactive IBV vaccine group, attenuated group comparison wasdiscovered extremely notable difference after the 14 day (P＜0.01),pDNAIL-1βplus IBV DNA group and all groups comparison was discoveredextremely notable difference after the 21 day (P＜0.01), in 28 days pDNAIL-1βplus IBV DNA group and IBV DNA group comparison appeared notabledifference; Outside cycle blood CD3+ T B lymphocyte B quantity that pDNAIL-1βplus IBV DNA was discovered extremely notable difference comparing withDNA vaccine group in 14 day (P＜0.01), notable difference pDNAIL-1βplus IBVDNA group and IBV DNA group comparison appeared in 21 day (P＜0.05). CD4+ thatpDNAIL-1βplus IBV DNA group and pcDNA3.1 group, inactive IBV vaccine group,attenuated group and PBS group comparison appeared extremely notabledifference(P＜0.05) in 7 days. pDNAIL-1βplus IBV DNA group and IBV DNA groupcomparison appeared extremely notable difference(P＜0.01) in 14-21 dayperiod. CD8+ that IBV DNA group with pDNAIL-1βand IBV DNA group comparisonappeared extremely notable difference(P＜0.05) in 14 day, In 21 days pDNAIL-1βplus IBV DNA group and pcDNA3, lgroup, pDNAIL-1βgroup, PBS group, inactiveIBV vaccine group, attenuated group appeared extremely notable difference(P＜0.05).
By the experiment of injection IBV to Gallus domesticus Brisson pDNAIL-1βplus IBV DNA group its protection ratio (95%) remarkably was higher thanthe independent IBV DNA vaccine group (85%) and the same with attenuated IBVvaccine's protection ratio, but lower than inactivated IBV vaccine'sprotection ratio.
We studied pDNAIL-1βby measuring CD3+,CD4+,CD8+, its protection ratioand indirect ELISA. IBV DNA vaccine displayed pDNAIL-1βcould enhanceimmunity function. We thought pDNAIL-1βwould be a very useful immuneintensifier in future.
用间接ELISA测定特异性抗体，结果表明，免疫14天时pDNAIL-1β+DNA组和弱毒苗组、灭活苗组比较差异极显著(P＜0.01)，在21天pDNAIL-1β+IBV DNA组与所有实验组比较差异极显著(P＜0.01)，在28天pDNAIL-1β+DNA组与DNA疫苗组比较差异显著(P＜0.05)；测定CD3+T淋巴细胞结果表明，pDNAIL-1β+IBV DNA组与IBV DNA疫苗组比较在14天差异极显著(P＜0.01)，21天时pDNAIL-1β+IBV DNA组与IBV DNA疫苗组比较差异显著(P＜0.05)，pDNAIL-1β+IBV DNA组与其它实验组比较均出现差异极显著(P＜0.01)；检测CD4+T淋巴细胞结果表明，7天时pDNAIL-1β+IBV DNA组与pcDNA3.1组、灭活苗组、弱毒苗组和PBS苗组比较差异显著(P＜0.05)，在14-21天期间pDNAIL-1β+IBV DNA组与IBV DNA疫苗组比较，差异极显著(P＜0.01)；研究CD8+结果表明，在免疫14天，pDNAIL-1β+IBV DNA组和IBV DNA疫苗组比较差异极显著(P＜0.01)，21天时与除DNA疫苗组外的其它五个实验组比较差异显著(P＜0.05)。
攻毒实验结果表明，pDNAIL-1β+IBV DNA组其死亡保护率(95％)显著高于IBV DNA疫苗组(85％)，与弱毒苗组的保护率相当(95％)，但低于灭活苗组的保护率(100％)。
综上表明pDNAIL-1β可以明显地增强IBV DNA疫苗的免疫作用，可作为一种有效的免疫增强剂。
We designed the primers based on chicken interleukin-1βin Genebank(Y15006),and then Gallus domesticus Brisson interleukin-1βwas amplifiedby recerse transcription-polymerase chain reaction(RT-PCR)from peripheralblood lymphocytes stimulated with ConA. The gene was cloned into PMD18-T vectorand then sequenced. Gallus domesticus Brisson IL-1βwas the fouth nucleotidesequence reported in the world. The result showed that the length of cDNA was804bp, encoding 267 amino acids, sharing 99.5% homology with the ChIL-1βgeneof Weining, K. C. in Genbank. The nucleotide sequence shared 99.0% and 97.9%homology with the ChIL-1βgene of Shao Weixing and Zhang Yongxia clonedrespectively. Wecarried on the homology analysis to those encoded amino acidand the evolution tree analysis 4 nucleotides sequences. Then digested thegene with BamHⅠand E_(CO)RⅠ, inserted into pcDNA3.1(+) which was also digestedwith BamHⅠand E_(CO)RⅠ. The eukaryatic expression vector of chickeninterleukin-1βgene (pDNAIL-1β) was constructed. By identified accuracypDNAIL-1βcould be used as an immune intensifier to immunity 7 groups ofchickens(pcDNA3. Igroup, IBV DNA group, pDNAIL-1βgroup, pDNAIL-1βplus IBVDNA group, PBS group, inactive IBV vaccine group, attenuated group).
With indirect ELISA admeasurement, peculiarity antibody of pDNAIL-1βplusIBV DNA group and inactive IBV vaccine group, attenuated group comparison wasdiscovered extremely notable difference after the 14 day (P＜0.01),pDNAIL-1βplus IBV DNA group and all groups comparison was discoveredextremely notable difference after the 21 day (P＜0.01), in 28 days pDNAIL-1βplus IBV DNA group and IBV DNA group comparison appeared notabledifference; Outside cycle blood CD3+ T B lymphocyte B quantity that pDNAIL-1βplus IBV DNA was discovered extremely notable difference comparing withDNA vaccine group in 14 day (P＜0.01), notable difference pDNAIL-1βplus IBVDNA group and IBV DNA group comparison appeared in 21 day (P＜0.05). CD4+ thatpDNAIL-1βplus IBV DNA group and pcDNA3.1 group, inactive IBV vaccine group,attenuated group and PBS group comparison appeared extremely notabledifference(P＜0.05) in 7 days. pDNAIL-1βplus IBV DNA group and IBV DNA groupcomparison appeared extremely notable difference(P＜0.01) in 14-21 dayperiod. CD8+ that IBV DNA group with pDNAIL-1βand IBV DNA group comparisonappeared extremely notable difference(P＜0.05) in 14 day, In 21 days pDNAIL-1βplus IBV DNA group and pcDNA3, lgroup, pDNAIL-1βgroup, PBS group, inactiveIBV vaccine group, attenuated group appeared extremely notable difference(P＜0.05).
By the experiment of injection IBV to Gallus domesticus Brisson pDNAIL-1βplus IBV DNA group its protection ratio (95%) remarkably was higher thanthe independent IBV DNA vaccine group (85%) and the same with attenuated IBVvaccine's protection ratio, but lower than inactivated IBV vaccine'sprotection ratio.
We studied pDNAIL-1βby measuring CD3+,CD4+,CD8+, its protection ratioand indirect ELISA. IBV DNA vaccine displayed pDNAIL-1βcould enhanceimmunity function. We thought pDNAIL-1βwould be a very useful immuneintensifier in future.
引文
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