【作者】 刘蕊娜；

【导师】 黄勇； 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 根据GenBank中鸡白介素6(ChIL-6)基因序列(AJ309540)设计一对引物，采用RT-PCR方法从ConA诱导的四川雅安土鸡脾淋巴细胞总RNA中扩增出约600bp的基因片段，克隆到pMD18-T载体，进行序列测定和分析。结果显示：该克隆的cDNA全长603bp，包含了鸡IL-6基因的开放阅读框585bp，编码195个氨基酸残基，与GenBank上仅有的1条鸡IL-6序列(AJ309540)的同源性为100％。生物信息学分析表明，该基因编码的蛋白具有亲水性，有很强的抗原性，有3个蛋白激酶C磷酸化位点，2个酪蛋白激酶Ⅱ磷酸化位点。该基因经Xho I和Xba I双酶切后，插入同样经Xho I和Xba I双酶切真核表达载体pCI-neo中，构建出真核表达质粒pCI-IL-6，通过免疫试验和攻毒试验观察其对新城疫LaSota疫苗的免疫增强作用。将21日龄非免疫健康雏鸡100只随机分为5组，肌肉免疫(LaSota疫苗组、pCI-IL-6+LaSota疫苗组、pCI-IL-6组、pCI-neo组和PBS组)。通过检测HI抗体、CD4~+、CD8~+和CD3~+T淋巴细胞百分含量，分析pCI-IL-6对LaSota疫苗的免疫佐剂效应。各组试验鸡在免疫接种后的不同时期，用血凝抑制试验(HI)测定NDV抗体，结果表明，pCI-IL-6和LaSota疫苗联合免疫组从第14天起就一直高于LaSota疫苗单独免疫组，在第14、21、35天时差异显著(P<0．05)，在28天时差异极显著(P<0．01)；pCI-IL-6和LaSota疫苗联合免疫组与其它试验组比较差异均极显著(P<0．01)。用流式细胞仪检测外周血代表T细胞亚群的CD4~+、CD8~+和CD3~+。CD4~+T淋巴细胞百分含量检测结果表明，pCI-IL-6+LaSota疫苗联合免疫组与LaSota疫苗单独免疫组相比，在14天出现差异显著(P<0．05)，在21、28天差异极显著(P<0．01)；pCI-IL-6+LaSota疫苗与其它试验组比较均差异极显著(P<0．01)。CD8~+T淋巴细胞百分含量检测结果表明，pCI-IL-6+LaSota疫苗联合免疫组和LaSota疫苗单独免疫组相比，在免疫后14天出现差异极显著(P<0．01)，第28天差异显著(P<0．05)；14天时与其它试验组比较均差异极显著(P<0．01)。CD3~+T淋巴细胞百分含量测定结果表明，LaSota疫苗联合佐剂pCI-IL-6组与单独LaSota疫苗组比较在7、28、35天差异显著(P<0．05)，在14、21天时差异极显著(P<0．01)；与其它试验组比较均差异极显著(P<0．01)。在免疫后35天进行攻毒，pCI-IL-6和LaSota疫苗联合免疫组的保护率为89．5％，而LaSota疫苗单独免疫组的保护率为76．5％。表明，pCI-IL-6能提高新城疫LaSota疫苗的免疫效果，能提高新城疫疫苗免疫的细胞和体液免疫水平及攻毒保护率。鸡IL-6真核质粒免疫佐剂效应的研究国内外尚未见报道。更多还原

【Abstract】 Two primers were designed and synthesized according to the chicken interleukin-6 sequence published in GenBank (AJ309540). The Gallus interleukin-6 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from splenocytes lymphocytes stimulated with ConA. The gene was inserted into pMD18-T vector and sequenced. The result showed that the length of this cDNA was 603bp, encoding 195 amino acids, and sharing 100% homology with the only one ChIL-6 sequnce (AJ309540) in GenBank. Bioinformatics analysis of this sequence shows that the protein of ChIL-6 was hydrophilic and highly antigenic, including three potential protein kinase C phosphor -ylation site, six potential Casein kinaseⅡphosphorylation site. The cDNA was digested with Xho /and XbaⅠand inserted into pCI-neo vector which was also digested with Xho /and Xba /to construct the eukaryotic expression vector pCI-IL-6. Then an immune experiment was designed to evaluate the immune adjuvant effect of pCI-IL-6.One hundred chickens were randomly assigned to five groups of twenty animals per treatment, including LaSota vaccine group, pCI-IL-6+LaSota vaccine group, pCI-IL-6 group, pCI-neo group and PBS control group. The immune adjuvant effect of the pCI-IL-6 to LaSota had been analyzed using the Hammaeglutination test (HI) antibody and the percentage of the CD4~+, CD8~+ and CD3~+T lymphocyte.The results shows that HI antibody of pCI-IL-6+LaSota vaccine group shows notable difference in 14,21, 35day (P<0.05) with LaSota vaccine group, shows extremely notable difference(P<0.01) in 28 days, HI antibody of pCI-IL-6+LaSota vaccine group shows extremely notable difference (P<0.01)with other treatments. The percentage of CD4~+, CD8~+ and CD3~+T lymphocyte was tested by flow cytometer technique, the results shows that the percentage of CD4~+T lymphocyte of pCI-IL-6 +LaSota vaccine group shows shows notable difference and extremely notable difference in 14 day (P<0.05) and 21, 28day (P<0.01), pCI-IL-6+LaSota vaccine group shows extremely notable difference in all days (P<0.01) with other groups. The percentage of CD8~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14 day (P<0.01) with LaSota vaccine group, shows notable difference in the 28 day (P<0.05). The percentage of CD3~+T lymphocyte of pCI-IL-6+LaSota vaccine group shows extremely notable difference in the 14, 21day (P<0.01) with LaSota vaccine group, shows notable difference in the 7, 28, 35day (P<0.05). Challenge test on 35th day after immunization shows that the protection ratio of co-immunization group of pCI-IL-6 and LaSota was 89.5%, while LaSota vaccine group was only 76.5%.The results indicated that pCI-IL-6 can improve the NDV immunity level of the humoral and cellualrimmunity as well as the challenge protection rate, showing immune adjuvant function. As we know, the immune adjuvant effect of eukaryotic plasmid of the ChIL-6 to LaSota vaccine hasn’t been reported now, this study provides referrence to the united application of pCI-IL-6 and NDV vaccine.更多还原