泰山松花粉多糖對(duì)禽波氏桿菌DNA疫苗的免疫增強(qiáng)作用
發(fā)布時(shí)間：2018-11-28 08:08
【摘要】：禽波氏桿菌病是一種新型細(xì)菌性傳染病,其病原體為禽波氏桿菌,主要造成雛禽的呼吸道疾病,引起急性死亡�；疾‰u群生長(zhǎng)緩慢,飼料報(bào)酬率低,給養(yǎng)禽業(yè)造成了巨大的經(jīng)濟(jì)損失。目前國(guó)內(nèi)外用于預(yù)防該病所用的疫苗主要有禽波氏桿菌溫度敏感株和全細(xì)胞菌素,但疫苗保護(hù)效果一般,僅能適當(dāng)降低病變的嚴(yán)重程度或延緩臨床癥狀的出現(xiàn),亟待研發(fā)新型疫苗。而DNA疫苗是一種最新的分子水平的生物技術(shù)疫苗,能夠同時(shí)誘導(dǎo)機(jī)體產(chǎn)生全面的體液免疫反應(yīng)和細(xì)胞免疫反應(yīng),因此在實(shí)踐應(yīng)用中備受人們關(guān)注。此外,本實(shí)驗(yàn)室從2003年開(kāi)始對(duì)泰山松花粉多糖(TPPPS)進(jìn)行了一系列的研究,發(fā)現(xiàn)泰山松花粉多糖作為疫苗佐劑能夠顯著提高滅活疫苗和亞單位疫苗的免疫效果,然而以泰山松花粉多糖作為DNA疫苗佐劑尚未被研究。因此,為研發(fā)新型有效的禽波氏桿菌疫苗,本課題選取禽波氏桿菌外膜蛋白A(OmpA)基因?yàn)槟康幕驑?gòu)建真核重組表達(dá)質(zhì)粒并制備DNA疫苗。為提高DNA疫苗的免疫效果,我們選取泰山松花粉多糖作為疫苗佐劑同時(shí)評(píng)估其對(duì)DNA疫苗的免疫調(diào)節(jié)作用。根據(jù)NCBI發(fā)表的禽波氏桿菌OmpA基因設(shè)計(jì)一對(duì)特異性引物,對(duì)實(shí)驗(yàn)室分離得到的禽波氏桿菌菌株的OmpA基因進(jìn)行PCR擴(kuò)增并連接到pMD18-T質(zhì)粒,轉(zhuǎn)化并送至測(cè)序公司測(cè)序。用EcoR I與Xho I酶切處理pMD18-T-OmpA質(zhì)粒獲得OmpA基因并將得到的OmpA基因連接到已用EcoR I與Xho I處理的pVAX1質(zhì)粒上,將重組質(zhì)粒進(jìn)行雙酶切檢測(cè)與測(cè)序。將驗(yàn)證構(gòu)建正確的重組真核質(zhì)粒pVAX1-OmpA轉(zhuǎn)染BHK-21細(xì)胞,72 h后收獲細(xì)胞上清液獲得表達(dá)的OmpA蛋白。將獲得的OmpA蛋白通過(guò)His標(biāo)簽鎳柱層析法進(jìn)行純化,純化的OmpA蛋白的SDS-PAGE分析顯示目的條帶大小約為27 KD,與預(yù)期結(jié)果相符。同時(shí),OmpA蛋白的Western blotting鑒定可清晰地看見(jiàn)約27 KD大小的特異性條帶,與其SDS-PAGE分析結(jié)果相一致。依照本實(shí)驗(yàn)室改進(jìn)的水提醇沉法提取泰山松花粉多糖,并將大量提取的重組質(zhì)粒與泰山松花粉多糖或弗氏不完全佐劑等體積配比制備疫苗。將224只1日齡SPF雛雞隨機(jī)分為7組,于1、7、14日齡時(shí),每組每只雛雞分別注射等量的3個(gè)不同多糖濃度(50 mg/mL、100 mg/mL、200 mg/mL)的DNA疫苗、弗氏佐劑DNA疫苗、純DNA疫苗、空質(zhì)粒和PBS。在首次免疫后的第7、14、21、28、35、42、49天每組隨機(jī)選取3只雛雞采血檢測(cè)其血清抗體效價(jià)、白細(xì)胞介素-2(IL-2)含量、白細(xì)胞介素-4(IL-4)含量、干擾素-γ(IFN-γ)含量,外周血CD4+和CD8+T淋巴細(xì)胞數(shù)量水平。同時(shí),在三次加強(qiáng)免疫后每組隨機(jī)選取20只進(jìn)行攻毒保護(hù)試驗(yàn),連續(xù)7天記錄雞群生存狀態(tài)并統(tǒng)計(jì)免疫保護(hù)率。結(jié)果顯示,禽波氏桿菌OmpA DNA疫苗能夠刺激機(jī)體產(chǎn)生特異性抗體、提高細(xì)胞因子分泌量和外周血CD4+和CD8+T淋巴細(xì)胞數(shù),并為雛雞提供50%的免疫保護(hù)。另外,泰山松花粉多糖和弗氏佐劑能夠顯著提高雛雞體液免疫和細(xì)胞免疫水平且200 mg/mL的松花粉多糖效果最優(yōu)(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- 緯 (IFN- 緯) 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- 緯 (IFN- 緯) 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).
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.12
本文編號(hào)：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- 緯 (IFN- 緯) 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- 緯 (IFN- 緯) 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).
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.12
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 宋成軍;龐曉靜;馬鶴超;;松花粉對(duì)D-半乳糖衰老大鼠肝臟抗氧化能力的影響[J];承德醫(yī)學(xué)院學(xué)報(bào);2006年03期
2 劉協(xié),張馳,包六行,潘玉英;松花粉對(duì)小鼠急性酒精性肝損傷的保護(hù)作用研究[J];江蘇預(yù)防醫(yī)學(xué);2005年02期
3 馬成亮;松花粉開(kāi)發(fā)利用價(jià)值的研究[J];林業(yè)科技;2002年05期
4 陳小燕;高澤立;;多糖的研究進(jìn)展—多糖對(duì)機(jī)體免疫功能的影響[J];胃腸病學(xué)和肝病學(xué)雜志;2006年05期
5 王玉燕,朱瑞良;禽波氏桿菌病原性研究及防治初探[J];中國(guó)預(yù)防獸醫(yī)學(xué)報(bào);2001年02期
6 朱瑞良，張紹學(xué)，唐珂心，牛鐘相;山雞波氏桿菌病的診斷[J];中國(guó)獸醫(yī)雜志;1994年01期
相關(guān)碩士學(xué)位論文 前1條
1 孟秀彥;泰山松花粉對(duì)雞兔免疫增強(qiáng)作用的研究[D];山東農(nóng)業(yè)大學(xué);2007年
，本文編號(hào)：2362303
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2362303.html
最近更新
教材專著
