CN102994520B - 鸡传染性法氏囊病vp2基因及其应用 - Google Patents
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Abstract
本发明涉及一种鸡传染性法氏囊病VP2基因，其氨基酸序列为SEQ ID NO:1。本发明筛选出的鸡传染性法氏囊病VP2基因用作基因工程亚单位疫苗。本发明筛选出的鸡传染性法氏囊病VP2基因制备的亚单位疫苗免疫21日龄SPF鸡，21日后能产生坚强免疫力，使免疫鸡抵抗鸡传染性法氏囊病病毒强毒的攻击。
Description
技术领域
本发明属于禽类抗原筛选技术领域，具体涉及一种鸡传染性法氏囊病VP2基因及其应用。
背景技术
鸡传染性法氏囊病（Infectious Bursal Disease，IBD）又称鸡传染性腔上囊病，是由传染性法氏囊病毒引起的一种急性、接触传染性疾病。以法氏囊发炎、坏死、萎缩和法氏囊内淋巴细胞严重受损为特征，能够引起鸡的免疫机能障碍，干扰各种疫苗的免疫效果。该病发病率高，是目前养禽业最重要的疾病之一。
接种疫苗是一种有效的预防传染性法氏囊病的措施，目前普遍使用的弱毒苗和灭活苗虽然对IBD的预防起了相当大的作用，但由于近年来鸡传染性法氏囊病毒（IBDV）变异株和超强毒株的出现，经常导致免疫失败，传统的灭活疫苗和弱毒疫苗已经面临着严峻的考验，基因工程亚单位疫苗的出现为鸡传染性法氏囊病预防提供了一条新的途径。
基因工程亚单位疫苗又称重组亚单位疫苗或生物合成亚单位疫苗，是利用DNA重组技术，将编码病原微生物保护性抗原的基因导入受体菌（如大肠杆菌）或细胞，使其在受体中高效表达，分泌保护性抗原肽链。提取保护性抗原肽链，加入佐剂即制成基因工程亚单位疫苗。但制备基因工程亚单位疫苗的关键是筛选出新的，具有更好免疫原性的抗原基因。
发明内容
本发明的目的是提供一种鸡传染性法氏囊病毒（IBDV）的主要宿主保护性抗原VP2基因，即一种具有了IBD超强毒的VP2基因，并将该基因重组表达后制成亚单位疫苗，从而有效弥补现有鸡传染性法氏囊病疫苗对IBDV变异株免疫不高的缺陷。
本发明筛选出的鸡传染性法氏囊病VP2基因，其氨基酸序列为SEQ IDNO:1。
上述鸡传染性法氏囊病VP2基因的核苷酸序列为SEQ ID NO:2。
本发明筛选出的鸡传染性法氏囊病VP2基因用作基因工程亚单位疫苗。
本发明筛选出的鸡传染性法氏囊病VP2基因制备的亚单位疫苗免疫21日龄SPF鸡，21日后能产生坚强免疫力，使免疫鸡抵抗鸡传染性法氏囊病病毒强毒的攻击。
具体实施方式
IBDV的主要结构蛋白VP2蛋白是最主要宿主保护性抗原，本发明是用PCR方法扩增了IBD超强毒的VP2基因，克隆到pET-28a载体上，转化BL21(DE3)，经IPTG诱导后VP2蛋白获得了表达，将表达菌体高压匀浆破碎后离心取上清即为VP2蛋白溶液，加入矿物油佐剂制成油乳剂疫苗，可用于预防鸡传染性法氏囊病。
下面结合具体的实施例对本发明的鸡传染性法氏囊病毒VP2基因进行详细的描述。
一、VP2基因的筛选
用Primer5.0软件设计了一对扩增鸡传染性法氏囊病病毒VP2全基因引物，并在上游引物5′端加入保护性碱基及BamH I酶切位点，在下游引物的5′端加入保护性碱基和Xho I酶切位点，引物序列为：
Primer 1：5′-GCG TCG ACA TGA CAA ACC TGC AAG ATC- 3′
Primer 2：5′-CCC TCG AGT TAT CCT TAT GGC CCG GATT -3′
取200 μl用临床分离的疑似IBDV的病料感染的鸡胚接种尿囊膜悬液上清液，按RNAisoReagent说明书所述方法提取病毒RNA，按反转录试剂盒使用说明书进行反转录合成cDNA， 进行PCR扩增，扩增体系为（50 μl）：10×PCR Buffer 5μl，dNTP (10mM each)1μl，上、下游引物(10mM)各 1μl， cDNA 3 μl， 25mM MgCl2 4μl， Taq酶 (5U/μl ) 1μl，ddH2O 34μl；PCR反应参数：95℃ 5min，95℃ 1min，58℃ 2min，72℃ 2min，35个循环，72℃ 10min 。得到一个1300bp左右的片段，切胶回收该片段，与pMD-18T载体连接。PCR回收片段6μl，T载体2μl (稀释10倍后)，T4DNA连接酶 1μl，T4 buffer 2.5μl，水:13.5μl，总计25μl。16℃过夜连接，取20 μl连接产物转化感受态大肠杆菌JM109，挑选10个白色菌落培养，提取质粒鉴定正确的选取3个测序，结果3个质粒的测序结果都一致，证明获得的核苷酸片段在扩增的过程中没有产生错误，其核苷酸序列为SEQ ID NO:2，翻译的氨基酸序列为SEQID NO:1。
将该核苷酸序列在NCBI上Blast比对，有十几个碱基发生了点突变，导致翻译的氨基酸序列也有几个位点发明变化。表明该毒株为一新的流行毒株。这是由于VP2蛋白是主要宿主保护性抗原，经常发生突变而导致抗原性发生差异。
二、 VP2重组蛋白的表达
将鉴定正确的上述一个质粒用BamH I和Xho I双酶切回收基因片段，与用这两个酶切的pET-28a载体连接，转化感受态大肠杆菌JM109，提取质粒鉴定正确后转化感受体的BL21（DE3），挑取单菌落，转接5mlLB小管培养基，37℃振荡培养3~4小时至OD600为0.6时，加入终浓度为0.8mM的IPTG诱导4~5小时，SDS-PAGE电泳结果证实为分泌表达。
将表达菌用10倍菌体湿重PBS重悬，高压匀浆破碎细菌，离心取上清，做琼脂免疫扩散试验，结果琼扩效价不低于1:64。
三、VP2蛋白的纯化
用镍柱纯化，200mM的咪唑洗脱，洗脱液用PBS透析过夜。所得透析液即为纯化的蛋白，可用于制苗。
四、VP2亚单位疫苗的制备
取注射用白油94份、司本-80 6份、硬脂酸铝2份，混合后，加热溶化， 116℃灭菌30分钟，作为制苗用油相；加入灭菌的吐温-80 4份，然后加入上述制备的VP2重组蛋白发酵提取液96份，充分振摇，使吐温-80完全溶解，作为制苗用水相；取油相3份放于胶体磨内，开动电机慢速转动搅拌，同时徐徐加入水相l份，加完后再以l0000r/min搅拌2～5分钟，在终止搅拌前加入1％硫柳汞溶液，使其最终浓度为0.01％。
VP2亚单位疫苗的免疫效果：
将20只SPF鸡随机分为两组，每组10只，其中一组在21日龄时以0.3ml/只的剂量皮下注射VP2亚单位疫苗，免疫21日后连同不免疫对照组每只经点眼途径接种100BID鸡传染性法氏囊病强毒BC6-85株毒液，攻毒后每天观察鸡只的临床表现及死亡情况，记录发病和死亡鸡数，剖检观察死亡鸡法氏囊等病变，72～96小时扑杀存活鸡，逐只剖解，观察法氏囊等病理变化。结果不免疫对照组8只鸡发病，而免疫组鸡只无发病。上述结果证明本发明制备的VP2亚单位疫苗具有很好的免疫原性。
Claims (3)
1.一种鸡传染性法氏囊病VP2基因，其氨基酸序列为SEQ ID NO:1。
2.一种核苷酸，所述的核苷酸用于编码权利要求1所述的鸡传染性法氏囊病VP2基因；其序列为SEQ ID NO:2。
3.权利要求1所述的鸡传染性法氏囊病VP2基因在制备基因工程亚单位疫苗中的应用。
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