本文選題：火雞皰疹病毒 + H9亞型禽流感病毒��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：H9N2亞型禽流感病毒(avian influenza virus,AIV)自1992年首次在我國(guó)雞群中被發(fā)現(xiàn)并報(bào)道以來(lái),迅速在各地流行,嚴(yán)重危害養(yǎng)禽業(yè)的發(fā)展。研究表明,H9N2亞型AIV不經(jīng)適應(yīng)即可感染人和哺乳動(dòng)物,也可為感染人的流感病毒提供內(nèi)部基因,具有重要的公共衛(wèi)生意義。在我國(guó),獨(dú)特的地理環(huán)境、養(yǎng)殖模式及活禽市場(chǎng)的存在為H9N2亞型AIV的傳播提供了有利條件,而免疫壓力更是病毒發(fā)生重組和變異的重要因素。通過(guò)對(duì)我國(guó)2010年-2015年間H9N2亞型AIV分離毒株的血凝素(HA)基因序列分析顯示,近年來(lái)的分離毒株主要集中于h9.4.2.5分支上,而常用的滅活疫苗的毒株并不屬于此分支,且滅活疫苗不能有效地誘導(dǎo)細(xì)胞免疫應(yīng)答和黏膜免疫應(yīng)答。因此,滅活疫苗的免疫保護(hù)率有時(shí)不高�；痣u皰疹病毒(herpesvirus of turkey,HVT)疫苗是預(yù)防雞馬立克氏病的常用疫苗,具有較多的復(fù)制非必需區(qū)可供外源基因的插入,被廣泛應(yīng)用于重組病毒活疫苗的構(gòu)建。本研究擬以HVTFC126疫苗株為載體,構(gòu)建表達(dá)h9.4.2.5分支H9N2亞型AIVHA的重組病毒,研制新型H9亞型AIV活疫苗。本研究通過(guò)PCR擴(kuò)增出h9.4.2.5分支H9亞型AIV(YZ1406毒株)的HA基因,與人巨細(xì)胞病毒早期啟動(dòng)子(CMV)和TKpolyA組成表達(dá)盒。將該表達(dá)盒插入到HVTFC126疫苗株的US2非必需區(qū)片段中,構(gòu)建轉(zhuǎn)移載體pHVT-H9HA。將pHVT-H9HA與表達(dá)EGFP基因的重組HVTFC126病毒(rHVT-EGFP)基因組DNA共轉(zhuǎn)染雞胚成纖維細(xì)胞(CEF),經(jīng)同源重組,將位于rHVT-EGFP US2區(qū)內(nèi)的EGFP標(biāo)記基因替換為HA基因表達(dá)盒,獲得重組病毒rHVT-H9HA。通過(guò)PCR、Southern-blot、Western-blot和間接免疫熒光試驗(yàn)對(duì)rHVT-H9HA進(jìn)行鑒定,并對(duì)其在CEF上的生長(zhǎng)特性進(jìn)行檢測(cè)。結(jié)果顯示:rHVT-H9HA中,HA基因表達(dá)盒已正確插入到US2復(fù)制非必需區(qū)內(nèi),并能成功表達(dá)出大小85kD左右的目的蛋白;同時(shí),rHVT-H9HA在CEF上表現(xiàn)出與HVTFC126疫苗毒株相同的復(fù)制水平。本研究對(duì)重組病毒rHVT-H9HA進(jìn)行了免疫效力的初步評(píng)價(jià)。將80只1日齡SPF雞隨機(jī)分成四組:rHVT-H9HA免疫組、滅活苗免疫組、攻毒對(duì)照組和空白對(duì)照組。其中rHVT-H9HA免疫組于1日齡經(jīng)頸部皮下接種5000空斑形成單位(PFU)的rHVT-H9HA,滅活苗免疫組于14日齡時(shí)經(jīng)肌肉注射0.25 mL/雞的禽流感病毒H9亞型滅活苗。在7、14、21、28日齡,分別通過(guò)血凝抑制試驗(yàn)(HI)檢測(cè)各組試驗(yàn)雞的血清中H9亞型AIV的抗體效價(jià)。除空白對(duì)照組外,其余各組于28日齡均采用點(diǎn)眼/滴鼻方式以108EID50/雞的YZ1406毒株攻毒。攻毒后的第3d、5d,采集喉頭、泄殖腔拭子,進(jìn)行病毒分離,檢測(cè)各組試驗(yàn)雞的排毒情況。結(jié)果顯示:rHVT-H9HA免疫組的試驗(yàn)雞在疫苗接種后抗體水平呈持續(xù)上升趨勢(shì);在攻毒后的第5d,病毒分離率顯著降低,而攻毒對(duì)照組病毒分離率較高,表明重組病毒rHVT-H9HA對(duì)H9亞型AIV的攻擊具有較好的保護(hù)作用。
[Abstract]:H9N2 subtype avian influenza virus (avian influenza, virus, AIV) since 1992 for the first time in chicken flocks in China were discovered and reported in the rapidly around the epidemic, serious harm to the poultry industry. The research results show that the H9N2 subtype of AIV without infection can adapt to human and mammal, can also provide internal genes for human infection the flu virus has important public health significance. In our country, the unique geographical environment, the spread of farming mode and live poultry market exists for the H9N2 subtype of AIV provided favorable conditions, but the immune pressure is an important factor of virus recombination and variation. Through to our country during the period of 2010 -2015, H9N2 subtype AIV isolates of hemagglutinin (HA) gene sequence analysis showed that the isolates in recent years mainly focused on h9.4.2.5 branch, and the commonly used inactivated vaccine strain does not belong to the branch, and the inactivated vaccine can effectively induce the fine The cellular immune response and mucosal immune response. Therefore, the immune protection of inactivated vaccine rate is not high. Sometimes the turkey herpes virus (herpesvirus of, Turkey, HVT) is a commonly used vaccine vaccine to prevent Marek's disease, has many nonessential regions can be inserted into the exogenous gene, is widely used in recombinant virus vaccine the construction. This study is intended to HVTFC126 vaccine strain as the carrier, to construct a recombinant virus expressing h9.4.2.5 branch of H9N2 subtype AIVHA, to develop a new type of H9 subtype AIV vaccine. This study was amplified by PCR h9.4.2.5 branch of H9 subtype AIV (YZ1406 strain) HA gene, and human cytomegalovirus early promoter (CMV) and TKpolyA expression box. The box is inserted into the HVTFC126 vaccine strain US2 non essential Fragment Expression transfer vector construction of pHVT-H9HA. recombinant HVTFC126 virus pHVT-H9HA and EGFP gene expression (rHVT-EGFP) genomic DNA were transferred to the Infected chicken embryo fibroblast (CEF), by homologous recombination, EGFP marker gene will be located in the US2 region of rHVT-EGFP to replace the HA gene cassette, the recombinant virus rHVT-H9HA. by PCR, Southern-blot, Western-blot and indirect immunofluorescence assay for identification of rHVT-H9HA, and to detect the CEF in the growth characteristics. The results showed rHVT-H9HA, HA gene expression cassette was correctly inserted into US2 nonessential regions, and can successfully express the size of about 85kD protein; at the same time, rHVT-H9HA showed the same level of replication and HVTFC126 vaccine strains in CEF. The recombinant virus in this study evaluated the immune effect rHVT-H9HA. 80 1 day old SPF chickens were randomly divided into four groups: rHVT-H9HA group, inactivated vaccine against the virus group, control group and blank control group. The rHVT-H9HA group at 1 days after subcutaneous inoculation of 5000 plaque Forming unit (PFU) rHVT-H9HA, inactivated vaccine group at the age of 14 days by intramuscular injection of 0.25 mL/ of chicken H9 subtype avian influenza virus inactivated vaccine. At 7,14,21,28 days, respectively by hemagglutination inhibition (HI) antibody titer of H9 subtype AIV in serum were detected in chickens. Except control group, other groups at 28 days of age were used to 108EID50/ nasal drops / chicken strains of YZ1406 virus attack. 3D, after infection of 5D, collecting the throat, cloacal swabs, virus isolation, detoxification detected chickens. The results showed that chickens immunized with rHVT-H9HA the antibody level after vaccination showed a rising trend; at 5D after infection, virus isolation rate is significantly reduced, and the challenge control group showed higher separation rate of the virus, the recombinant virus of rHVT-H9HA subtype H9 AIV attack has a good protective effect.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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