【作者】 张毅；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 采用PCR从含有鸡白细胞介素1β(interleukin-1β，IL-1β)与白细胞介素16(interleukin-16，IL-16)基因的PMD18-T质粒PCIL-1β、PCIL-16中获取鸡IL-1β与IL-16基因，分别用BamHⅠ和EcoRⅠ、BamHⅠ和EcoRⅤ双酶切后与同样酶切过的真核表达质粒pcDNA3.1连接，并进行序列测定，pDNAIL-1β中IL-1β基因全长开放阅读框为804bp，pDNAIL-16中IL-16基因全长开放阅读框为1824bp，与PCIL-1β、EIL-16中IL-1β与IL-16序列完全一致，成功构建了pDNAIL-1β及pDNAIL-16真核表达质粒。将两种质粒用阳离子脂质体包裹，分别或共同与禽流感疫苗联合免疫，观察IL-1β、IL-16分子佐剂对H5亚型禽流感灭活疫苗的佐剂效应。将1日龄非免疫健康雏鸡100只，分成5组：重组H5亚型禽流感病毒灭活疫苗AI(A)、AI-pcDNA3.1(B)、AI-pDNAIL-1β(C)、AI-pDNAIL-16(D)、AI-pDNAIL-1β+pDNAIL-16(E)。采用微量血凝抑制试验法(HI)，分别在免疫后7d、14d、21d、28d、35d检测禽流感特异性抗体，比较各组试验鸡的抗体水平。实验结果表明：与对照组相比，IL-1β组抗体水平显著提高(P＜0.05)，使抗体滴度较对照组提高了21g2以上，且在免疫后35d仍保持在接近峰值的水平。IL-16组抗体水平在实验前期迅速提高(P＜0.05)，但在实验中后期的HI抗体水平与对照组无显著差异(P＞0.05)。IL-1β+IL-16组HI抗体水平在免疫后迅速升高，并在免疫后14d显著高于IL-1β组与IL-16组(P＜0.05)，直到35d未见下降趋势。表明鸡IL-1β、IL-16分子佐剂可使禽流感疫苗免疫后HI抗体提前(免疫后7d)达到完全保护水平(≥51g2)，且明显延长抗体峰值的持续时间。用流式细胞仪对CD4+、CD8+及CD3+T细胞亚群进行了监测，实验结果表明：3个免疫佐剂组免疫后外周血中CD4+与CD3+T细胞的百分含量显著提高(P＜0.05)，IL-1β+IL-16组在实验后期CD4+T细胞百分含量高于IL-1β组和IL-16组，但差异不显著(P＞0.05)；CD3+T细胞亚群的变化与IL-1β组和IL-16组相近。分子佐剂组的CD8+T细胞水平与对照组无显著差异。表明鸡IL-1β、IL-16分子佐剂能显著提高机体CD4+、CD3+T细胞百分含量。本研究结果表明，表明鸡IL-1β、IL-16分子佐剂可使禽流感疫苗免疫后HI抗体提前(免疫后7d)达到完全保护水平(≥51g2)，延长抗体峰值的持续时间，且显著提高CD4+、CD3+T细胞百分含量(P＜0.05)。鸡IL-1β、IL-16分子佐剂对H5亚型禽流感灭活疫苗的佐剂效应的研究国内未见报道，本研究为增强禽流感灭活疫苗的免疫应答水平的研究提供了一些参考。更多还原

【Abstract】 In this study, chicken interleukin-1βgene and interleukin-16 gene were cloned from PCIL-1βand PCIL-16. After digestion by BamHⅠ/EcoRⅠ、BamHⅠ/EcoR V, IL—1βand IL-16 were inserted into eukaryotic expression plasmid pcDNA3. 1 respectively. The inserts were confirmed by sequencing, and the ORF of IL-1βwas 804bp, while that of IL-16 was 1824bp. pDNAIL-1βand pDNAIL-16 were successfully constructed.We simultaneously immunized chicken with H5 subtype AIV inactivated vaccine and pDNAIL-1β/pDNAIL-16 respectively or together. The immunological enhancing effects of molecular adjuvants IL-1βand IL-16 to H5 subtype AIV vaccine was investigated.100 unimmunized chicken(1-day-old) were devided to 5 groups: AI vaccine(A), AI-pcDNA3.1(B), AI-pDNAIL-1β(C), AI-pDNAIL-16(D), AI-pDNAIL-1β+pDNAIL-16(E). With Hemagglutination Inhibition(HI) test, sera collected 7, 14, 21, 28, 35d after inoculation, specific antibody was detected. The result indicated that IL-1β, IL-16, IL-1β+IL-16group reached 100% protection antibody level(≥51g2) 7d earlier than control groups. The anitbody level of IL-1βgroup significantly boosted at the middle and later stage of the experiment and kept it at a high level(P<0.05). The anitbody level of IL-16 group boosted at the early stage rapidly, but showed insignificant difference with control groups(P>0.05) at later stage. The anitbody level of IL-1β+IL-16 group approached the peak 14d post inoculation and significantly higher than IL-1βgroup and IL-16.The quantities of T cell subtypes were detected by flow cytometer. The CD4+ and CD3+T cell quantity of the 3 adjuvant groups significantly boosted(P<0.05), The CD4+ T cell quantity of IL-1β+IL-16 group was higher than IL-1βand IL-16 group, but the difference was insignificant(P>0.05). The CD3+T cell quantity of IL-1β+IL-16 group was similar to IL-1βand IL-16 group. As for the CD8+T cell quantity, the differences among all groups were insignificant at all stages of the experiment.The results indicated that chicken interleukin-1βand interleukin-16 molecular adjuvants were able to make the specific antibody reach 100% protective level(≥51g2) earlier than control group, prolong the peak period, and boost cellullar immunity of chicken provoked by H5 subtype AIV inactivated vaccine(P<0.05). This study had provided references to further research on enhancing the immune response of AIV vaccine with cytokins.更多还原