表达H9亚型禽流感病毒血凝素的重组火鸡疱疹病毒构建及其免疫效力试验

发布时间：2018-04-14 08:44

本文选题：火鸡疱疹病毒 + H9亚型禽流感病毒　；参考：《扬州大学》2017年硕士论文

【摘要】：H9N2亚型禽流感病毒(avian influenza virus,AIV)自1992年首次在我国鸡群中被发现并报道以来,迅速在各地流行,严重危害养禽业的发展。研究表明,H9N2亚型AIV不经适应即可感染人和哺乳动物,也可为感染人的流感病毒提供内部基因,具有重要的公共卫生意义。在我国,独特的地理环境、养殖模式及活禽市场的存在为H9N2亚型AIV的传播提供了有利条件,而免疫压力更是病毒发生重组和变异的重要因素。通过对我国2010年-2015年间H9N2亚型AIV分离毒株的血凝素(HA)基因序列分析显示,近年来的分离毒株主要集中于h9.4.2.5分支上,而常用的灭活疫苗的毒株并不属于此分支,且灭活疫苗不能有效地诱导细胞免疫应答和黏膜免疫应答。因此,灭活疫苗的免疫保护率有时不高。火鸡疱疹病毒(herpesvirus of turkey,HVT)疫苗是预防鸡马立克氏病的常用疫苗,具有较多的复制非必需区可供外源基因的插入,被广泛应用于重组病毒活疫苗的构建。本研究拟以HVTFC126疫苗株为载体,构建表达h9.4.2.5分支H9N2亚型AIVHA的重组病毒,研制新型H9亚型AIV活疫苗。本研究通过PCR扩增出h9.4.2.5分支H9亚型AIV(YZ1406毒株)的HA基因,与人巨细胞病毒早期启动子(CMV)和TKpolyA组成表达盒。将该表达盒插入到HVTFC126疫苗株的US2非必需区片段中,构建转移载体pHVT-H9HA。将pHVT-H9HA与表达EGFP基因的重组HVTFC126病毒(rHVT-EGFP)基因组DNA共转染鸡胚成纤维细胞(CEF),经同源重组,将位于rHVT-EGFP US2区内的EGFP标记基因替换为HA基因表达盒,获得重组病毒rHVT-H9HA。通过PCR、Southern-blot、Western-blot和间接免疫荧光试验对rHVT-H9HA进行鉴定,并对其在CEF上的生长特性进行检测。结果显示:rHVT-H9HA中,HA基因表达盒已正确插入到US2复制非必需区内,并能成功表达出大小85kD左右的目的蛋白;同时,rHVT-H9HA在CEF上表现出与HVTFC126疫苗毒株相同的复制水平。本研究对重组病毒rHVT-H9HA进行了免疫效力的初步评价。将80只1日龄SPF鸡随机分成四组:rHVT-H9HA免疫组、灭活苗免疫组、攻毒对照组和空白对照组。其中rHVT-H9HA免疫组于1日龄经颈部皮下接种5000空斑形成单位(PFU)的rHVT-H9HA,灭活苗免疫组于14日龄时经肌肉注射0.25 mL/鸡的禽流感病毒H9亚型灭活苗。在7、14、21、28日龄,分别通过血凝抑制试验(HI)检测各组试验鸡的血清中H9亚型AIV的抗体效价。除空白对照组外,其余各组于28日龄均采用点眼/滴鼻方式以108EID50/鸡的YZ1406毒株攻毒。攻毒后的第3d、5d,采集喉头、泄殖腔拭子,进行病毒分离,检测各组试验鸡的排毒情况。结果显示:rHVT-H9HA免疫组的试验鸡在疫苗接种后抗体水平呈持续上升趋势;在攻毒后的第5d,病毒分离率显著降低,而攻毒对照组病毒分离率较高,表明重组病毒rHVT-H9HA对H9亚型AIV的攻击具有较好的保护作用。
[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.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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