泰山松花粉多糖对禽波氏杆菌DNA疫苗的免疫增强作用
发布时间：2018-11-28 08:08
【摘要】：禽波氏杆菌病是一种新型细菌性传染病,其病原体为禽波氏杆菌,主要造成雏禽的呼吸道疾病,引起急性死亡。患病鸡群生长缓慢,饲料报酬率低,给养禽业造成了巨大的经济损失。目前国内外用于预防该病所用的疫苗主要有禽波氏杆菌温度敏感株和全细胞菌素,但疫苗保护效果一般,仅能适当降低病变的严重程度或延缓临床症状的出现,亟待研发新型疫苗。而DNA疫苗是一种最新的分子水平的生物技术疫苗,能够同时诱导机体产生全面的体液免疫反应和细胞免疫反应,因此在实践应用中备受人们关注。此外,本实验室从2003年开始对泰山松花粉多糖(TPPPS)进行了一系列的研究,发现泰山松花粉多糖作为疫苗佐剂能够显著提高灭活疫苗和亚单位疫苗的免疫效果,然而以泰山松花粉多糖作为DNA疫苗佐剂尚未被研究。因此,为研发新型有效的禽波氏杆菌疫苗,本课题选取禽波氏杆菌外膜蛋白A(OmpA)基因为目的基因构建真核重组表达质粒并制备DNA疫苗。为提高DNA疫苗的免疫效果,我们选取泰山松花粉多糖作为疫苗佐剂同时评估其对DNA疫苗的免疫调节作用。根据NCBI发表的禽波氏杆菌OmpA基因设计一对特异性引物,对实验室分离得到的禽波氏杆菌菌株的OmpA基因进行PCR扩增并连接到pMD18-T质粒,转化并送至测序公司测序。用EcoR I与Xho I酶切处理pMD18-T-OmpA质粒获得OmpA基因并将得到的OmpA基因连接到已用EcoR I与Xho I处理的pVAX1质粒上,将重组质粒进行双酶切检测与测序。将验证构建正确的重组真核质粒pVAX1-OmpA转染BHK-21细胞,72 h后收获细胞上清液获得表达的OmpA蛋白。将获得的OmpA蛋白通过His标签镍柱层析法进行纯化,纯化的OmpA蛋白的SDS-PAGE分析显示目的条带大小约为27 KD,与预期结果相符。同时,OmpA蛋白的Western blotting鉴定可清晰地看见约27 KD大小的特异性条带,与其SDS-PAGE分析结果相一致。依照本实验室改进的水提醇沉法提取泰山松花粉多糖,并将大量提取的重组质粒与泰山松花粉多糖或弗氏不完全佐剂等体积配比制备疫苗。将224只1日龄SPF雏鸡随机分为7组,于1、7、14日龄时,每组每只雏鸡分别注射等量的3个不同多糖浓度(50 mg/mL、100 mg/mL、200 mg/mL)的DNA疫苗、弗氏佐剂DNA疫苗、纯DNA疫苗、空质粒和PBS。在首次免疫后的第7、14、21、28、35、42、49天每组随机选取3只雏鸡采血检测其血清抗体效价、白细胞介素-2(IL-2)含量、白细胞介素-4(IL-4)含量、干扰素-γ(IFN-γ)含量,外周血CD4+和CD8+T淋巴细胞数量水平。同时,在三次加强免疫后每组随机选取20只进行攻毒保护试验,连续7天记录鸡群生存状态并统计免疫保护率。结果显示,禽波氏杆菌OmpA DNA疫苗能够刺激机体产生特异性抗体、提高细胞因子分泌量和外周血CD4+和CD8+T淋巴细胞数,并为雏鸡提供50%的免疫保护。另外,泰山松花粉多糖和弗氏佐剂能够显著提高雏鸡体液免疫和细胞免疫水平且200 mg/mL的松花粉多糖效果最优(P0.05)。
[Abstract]:Botrytis pneumoniae is a new type of bacterial infectious disease, which is mainly caused by respiratory tract diseases of young birds and causes acute death. The sick flocks grow slowly, feed rate of return is low, feed poultry industry has caused huge economic loss. At present, the main vaccines used to prevent the disease at home and abroad include thermo-sensitive strains and whole-cell bacteriocin, but the protective effect of the vaccine is general, which can only reduce the severity of disease or delay the emergence of clinical symptoms. New vaccines need to be developed. However, DNA vaccine is a new kind of biotechnological vaccine at molecular level, which can induce both humoral and cellular immune responses, so it has attracted much attention in practice. In addition, a series of studies on Taishan pine pollen polysaccharide (TPPPS) have been carried out in our laboratory since 2003. It was found that Taishan pine pollen polysaccharide as a vaccine adjuvant could significantly improve the immune effect of inactivated vaccine and subunit vaccine. However, the use of pine pollen polysaccharides as DNA vaccine adjuvant has not been studied. Therefore, in order to develop a new and effective vaccine, the recombinant eukaryotic expression plasmid was constructed by selecting the A (OmpA) gene as the target gene and the DNA vaccine was prepared. In order to improve the immune effect of DNA vaccine, we selected pine pollen polysaccharide as vaccine adjuvant and evaluated its immunomodulatory effect on DNA vaccine. According to the OmpA gene published by NCBI, a pair of specific primers were designed. The OmpA gene was amplified by PCR and ligated to pMD18-T plasmid, then transformed to sequencing company for sequencing. The OmpA gene was obtained by digesting pMD18-T-OmpA plasmid with EcoR I and Xho I, and the obtained OmpA gene was ligated to pVAX1 plasmid treated with EcoR I and Xho I. the recombinant plasmid was detected by double digestion and sequenced. The recombinant eukaryotic plasmid pVAX1-OmpA was constructed and transfected into BHK-21 cells. After 72 hours, the expressed OmpA protein was obtained from the supernatant of the cells. The purified OmpA protein was purified by His label nickel column chromatography. The SDS-PAGE analysis of the purified OmpA protein showed that the target band size was about 27 KD,. At the same time, the specific bands of about 27 KD can be seen clearly by Western blotting identification of OmpA protein, which is consistent with the results of SDS-PAGE analysis. The pollen polysaccharides of Pinus tabulaeformis were extracted by the improved water extraction and alcohol precipitation method in our laboratory. A large number of recombinant plasmids were mixed with the pollen polysaccharides or Freund's incomplete adjuvants of Pinus tabulaeformis to prepare the vaccine. Two hundred and twenty-four 1-day-old SPF chicks were randomly divided into 7 groups. At the age of 714 days, each chicks were injected with the same amount of DNA vaccine with different polysaccharides concentration (50 mg/mL,100 mg/mL,200 mg/mL) and Freund's adjuvant DNA vaccine. Pure DNA vaccine, empty plasmid and PBS. The serum antibody titer, interleukin-2 (IL-2) content, interleukin-4 (IL-4) content, interferon- 纬 (IFN- 纬) content were measured in 3 chicks in each group on day 7, 14, 21, 28, 35, 42, 49 days after the first immunization, and the serum antibody titer, interleukin-2 (IL-2) content, interleukin-4 (IL-4) content and interferon- 纬 (IFN- 纬) content were determined. The levels of CD4 and CD8 T lymphocytes in peripheral blood. At the same time, after three times of booster immunization, 20 chickens in each group were selected randomly to carry on the drug attack protection test, and the survival state of the chickens was recorded for 7 days and the immune protection rate was counted. The results showed that the OmpA DNA vaccine could stimulate the production of specific antibodies, increase the secretion of cytokines and the number of CD4 and CD8 T lymphocytes in peripheral blood, and provide 50% immunity protection for chicks. In addition, Taishan pine pollen polysaccharide and Freund's adjuvant could significantly improve the humoral and cellular immunity of chicks, and the effect of 200 mg/mL pine pollen polysaccharides was the best (P0.05).
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.12
本文编号：2362303
[Abstract]:Botrytis pneumoniae is a new type of bacterial infectious disease, which is mainly caused by respiratory tract diseases of young birds and causes acute death. The sick flocks grow slowly, feed rate of return is low, feed poultry industry has caused huge economic loss. At present, the main vaccines used to prevent the disease at home and abroad include thermo-sensitive strains and whole-cell bacteriocin, but the protective effect of the vaccine is general, which can only reduce the severity of disease or delay the emergence of clinical symptoms. New vaccines need to be developed. However, DNA vaccine is a new kind of biotechnological vaccine at molecular level, which can induce both humoral and cellular immune responses, so it has attracted much attention in practice. In addition, a series of studies on Taishan pine pollen polysaccharide (TPPPS) have been carried out in our laboratory since 2003. It was found that Taishan pine pollen polysaccharide as a vaccine adjuvant could significantly improve the immune effect of inactivated vaccine and subunit vaccine. However, the use of pine pollen polysaccharides as DNA vaccine adjuvant has not been studied. Therefore, in order to develop a new and effective vaccine, the recombinant eukaryotic expression plasmid was constructed by selecting the A (OmpA) gene as the target gene and the DNA vaccine was prepared. In order to improve the immune effect of DNA vaccine, we selected pine pollen polysaccharide as vaccine adjuvant and evaluated its immunomodulatory effect on DNA vaccine. According to the OmpA gene published by NCBI, a pair of specific primers were designed. The OmpA gene was amplified by PCR and ligated to pMD18-T plasmid, then transformed to sequencing company for sequencing. The OmpA gene was obtained by digesting pMD18-T-OmpA plasmid with EcoR I and Xho I, and the obtained OmpA gene was ligated to pVAX1 plasmid treated with EcoR I and Xho I. the recombinant plasmid was detected by double digestion and sequenced. The recombinant eukaryotic plasmid pVAX1-OmpA was constructed and transfected into BHK-21 cells. After 72 hours, the expressed OmpA protein was obtained from the supernatant of the cells. The purified OmpA protein was purified by His label nickel column chromatography. The SDS-PAGE analysis of the purified OmpA protein showed that the target band size was about 27 KD,. At the same time, the specific bands of about 27 KD can be seen clearly by Western blotting identification of OmpA protein, which is consistent with the results of SDS-PAGE analysis. The pollen polysaccharides of Pinus tabulaeformis were extracted by the improved water extraction and alcohol precipitation method in our laboratory. A large number of recombinant plasmids were mixed with the pollen polysaccharides or Freund's incomplete adjuvants of Pinus tabulaeformis to prepare the vaccine. Two hundred and twenty-four 1-day-old SPF chicks were randomly divided into 7 groups. At the age of 714 days, each chicks were injected with the same amount of DNA vaccine with different polysaccharides concentration (50 mg/mL,100 mg/mL,200 mg/mL) and Freund's adjuvant DNA vaccine. Pure DNA vaccine, empty plasmid and PBS. The serum antibody titer, interleukin-2 (IL-2) content, interleukin-4 (IL-4) content, interferon- 纬 (IFN- 纬) content were measured in 3 chicks in each group on day 7, 14, 21, 28, 35, 42, 49 days after the first immunization, and the serum antibody titer, interleukin-2 (IL-2) content, interleukin-4 (IL-4) content and interferon- 纬 (IFN- 纬) content were determined. The levels of CD4 and CD8 T lymphocytes in peripheral blood. At the same time, after three times of booster immunization, 20 chickens in each group were selected randomly to carry on the drug attack protection test, and the survival state of the chickens was recorded for 7 days and the immune protection rate was counted. The results showed that the OmpA DNA vaccine could stimulate the production of specific antibodies, increase the secretion of cytokines and the number of CD4 and CD8 T lymphocytes in peripheral blood, and provide 50% immunity protection for chicks. In addition, Taishan pine pollen polysaccharide and Freund's adjuvant could significantly improve the humoral and cellular immunity of chicks, and the effect of 200 mg/mL pine pollen polysaccharides was the best (P0.05).
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.12
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