一种牛呼吸道胞合体病毒抗原蛋白的制作方法

本发明属于生物产品制备
技术领域：
，涉及一种牛呼吸道胞合体病毒抗原蛋白及其制备方法。
背景技术：
：牛呼吸道胞合体病毒（BovineRespirationSyncytialVirus–BRSV）感染可引起牛呼吸道疾病，在欧共体国家被列为仅次于牛粘膜病（BVD）及牛传染性鼻气管炎（IBR）的重要牛病之一。本病呈世界范围性流行，其危害性在于BRSV感染发病率高达到60%-80%，死亡率在一些爆发中达到20%以上，给养牛业造成很大的经济损失。虽然这种BRSV从上个世纪70年代就开始列入研发范畴，由于种种原因并没有开发出理想的药物和疫苗。尽管已有关于BRSV的多种灭活，弱毒，重组以及DNA疫苗的报道，但距离临床使用还有一定距离，目前国际上尚没有理想的病毒疫苗。传统疫苗的研制和生产主要是通过改变培养条件，或在不同寄主动物上传代使致病微生物毒性减弱，或通过物理、化学方法将其灭活来完成的。随着人类知识的不断进步，传统疫苗的局限性也日益显露出来：(1)动物和人类的病毒需要在动物细胞中培养，这使得疫苗生产的成本很高；(2)疫苗中的致病物质在疫苗生产过程中有可能没有完全杀死或充分减毒，这会导致疫苗中含有强毒性致病物质，进而使得疾病在更大的范围内传播；(3)减毒菌株有可能会发生突变，引起病害。施一公开创了中国结构生物学基础理论研究的先河，本发明将结构生物学应用到实际中，合成牛呼吸道胞合体病毒抗原蛋白。牛呼吸道胞合体病毒是副黏液病毒科家族的成员，其上的F-蛋白是高度保守的并形成三聚体刺突，其在激活下发生构象变化。F-蛋白介导病毒和细胞膜的融合，让病毒核壳体进入细胞质，抑制F-蛋白介导的步骤阻抑感染周期的起始阶段并中和病毒感染性可以有效的防止病毒侵害，即针对F-蛋白的抗体，抑制它的活性，可以中和病毒感染性并可以保护免受BRSV感染。施一公开创了中国结构生物学基础理论研究的先河，本发明将结构生物学应用到实际中，合成牛呼吸道胞合体病毒抗原蛋白。技术实现要素：本发明的目的在于克服现有技术存在的缺点，提供一种牛呼吸道胞合体病毒抗原蛋白及其制备方法，该抗原蛋白应用蛋白质晶体学的技术和理论，确定蛋白在病毒侵染和致病过程中的结构变化，再根据结构变化的结果对这个关键的病毒蛋白进行基因工程改造，使其成为只侵染不致病、并引起动物体抗体高效反应的蛋白抗原。为了实现上述目的，本发明解决其技术问题所采取的技术方案是：一种牛呼吸道胞合体病毒抗原蛋白，编码牛呼吸道胞合体病毒关键蛋白F蛋白野生种的氨基酸序列简称SEQCAA76980.1，具体序列为MATTAMTMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQSLMQNEPASFSRAKRSIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISNIATVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLGKDQLSGINNLSFSK，其特征在于：所述牛呼吸道胞合体病毒抗原蛋白的氨基酸序列相比SEQCAA76980.1至少有两个位点被半胱氨酸替代，且替代后的半胱氨酸之间由二硫键连接。作为优化的，所述位点为氨基酸序列第148位缬氨酸和288位异亮氨酸。作为优化的，所述位点为氨基酸序列第158位亮氨酸和290位丝氨酸。作为优化的，所述牛呼吸道胞合体病毒抗原蛋白的氨基酸序列相比SEQCAA76980.1，在第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充。作为优化的，所述针对牛呼吸道胞合体病毒抗原蛋白的氨基酸序列剔除SEQCAA76980.1序列中的103位点至138位点的氨基酸序列，采用sgsgs连接102位点和139位点。作为优化的，所述针对牛呼吸道胞合体病毒抗原蛋白的氨基酸序列剔除SEQCAA76980.1序列中的103位点至142位点的氨基酸序列，采用sgsgs连接102位点和143位点。作为优化的，所述编码牛呼吸道胞合体病毒抗原蛋白的氨基酸序列相比SEQCAA76980.1序列，第99位点天冬氨酸由半胱氨酸替代，第362位点丝氨酸由半胱氨酸替代，并在替代后的半胱氨酸之间连接内二硫键。一种牛呼吸道胞合体病毒抗原蛋白的制备方法，其特征在于：具体工艺如下：（1）根据结构生物学的原理，进行蛋白结晶，结晶后用X-射线观测，确定侵染病毒关键病原F蛋白侵染前后的结构变化；（2）根据步骤（1）结构变化设计出抗原蛋白的序列和结构；（3）经多种抗原测试和稳定性特性检测确定出需要抗原蛋白的合格性；（4）将经过验证的抗原蛋白序列进行相应的蛋白合成、表达、提纯和检测，得到用来作为动物试验和临床试验的针对牛呼吸道胞合体病毒抗原蛋白。牛胞合体病毒关键F-蛋白介导病毒和细胞膜的融合，让病毒核壳体进入细胞。针对牛呼吸道胞合体病毒设计的抗原蛋白，便是根据F-蛋白侵染前后的结构变化设定的，在基因工程的基础上使F蛋白结构始终保持在融合前的状态，使其具有只侵染不致病、并引起动物体抗体高效反应的蛋白抗原等特点。本发明能够使F蛋白在侵染前后都保持三聚体刺突状态（闭合状态），得到只侵染不致病、并引起动物抗体高效反应的蛋白抗原；为了保证来自于病原病毒上的F-蛋白处在稳定的侵染前的状态，给F-蛋白上增添了系列的连接链，包括内二硫键、二硫键、空穴填充和单链连接。本发明与现有技术相比，其有益效果是：采用蛋白质晶体学方法制备的牛呼吸道胞合体病毒抗原蛋白能够有效的预防和治疗牛呼吸道胞合体病毒感染，以此为基础可以开发出理想的病毒疫苗；有效控制和防范病毒感染带来的损失，具有重大的经济意义和社会意义。附图说明图1本发明涉及利用Phenix软件进行牛合胞体病毒F-蛋白的三维立体结构模拟图，左图为浸染前三聚体三维立体结构模拟图，右图为左图右下角方框位置的局部放大图。图2本发明涉及的牛合胞体病毒呼吸道病毒侵染前后过程的关键的F-蛋白结构变化示意图。在图中显示侵染前（左），F蛋白三聚体呈现三聚体刺突状态，侵染后（右）F-蛋白被激活，发生构象变化，病毒和细胞膜的融合，病毒核壳体进入细胞质，进而侵染牛体并使其致病。图3本发明涉及的经过改造成功的牛呼吸道胞合体蛋白抗原侵染牛体后的电镜照片状态。左图显示RD-BRSV-DB1-CF1改造的牛呼吸道胞合体蛋白抗原侵染牛体后的状态，可见呈现稳定的融合前三聚体刺突状态，右图为未经过改造的牛呼吸道胞合体病毒侵染牛体后的电镜照片。图4本发明涉及的F-蛋白上添加连接链示意图，包括内二硫键、二硫键、空穴填充和单链连接，这些链接保证F-蛋白保持三聚体刺突状态。具体实施方式下面通过实施例并结合附图对本发明进一步说明。实施例1：将SEQCAA76980.1进行如下改造得到本发明的一种牛呼吸道胞合体病毒抗原蛋白：牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第148位缬氨酸由半胱氨酸替代，第288位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，将其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1，其氨基酸序列简称为SEQDB1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第158位亮氨酸由半胱氨酸替代，第290位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，将其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2，其氨基酸序列简称为SEQDB2。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第148位缬氨酸由半胱氨酸替代，第288位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充，将其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1，其氨基酸序列简称为SEQDB1-CF1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第158位亮氨酸由半胱氨酸替代，第290位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充，将其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1，其氨基酸序列简称为SEQDB2-CF1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第148位缬氨酸由半胱氨酸替代，第288位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接；同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充；剔除SEQCAA76980.1序列中103至位点138位点的氨基酸序列，采用sgsgs连接102及139位点，将其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1-SC1，其氨基酸序列简称为SEQDB1-CF1-SC1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第158位亮氨酸由半胱氨酸替代，第290位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充；剔除SEQCAA76980.1序列中103至位点138位点的氨基酸序列，采用sgsgs连接102及139位点，将其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1-SC1，其氨基酸序列简称为SEQDB2-CF1-SC1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第148位缬氨酸由半胱氨酸替代，第288位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接；同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充；剔除SEQCAA76980.1序列中103至位点138位点的氨基酸序列，采用sgsgs连接102及139位点，第99位点天冬氨酸由半胱氨酸替代，第362位点丝氨酸由半胱氨酸替代，并在替代后的半胱氨酸之间连接内二硫键，进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1-SC1-ID1，其氨基酸序列简称为SEQDB1-CF1-SC1-ID1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第148位缬氨酸由半胱氨酸替代，第288位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接；同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充；剔除SEQCAA76980.1序列中103至位点142位点的氨基酸序列，采用sgsgs连接102及143位点，第99位点天冬氨酸由半胱氨酸替代，第362位点丝氨酸由半胱氨酸替代，并在替代后的半胱氨酸之间连接内二硫键，进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB1-CF1-SC2-ID1，其氨基酸序列简称为SEQDB1-CF1-SC2-ID1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第158位亮氨酸由半胱氨酸替代，第290位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充；剔除SEQCAA76980.1序列中103至位点138位点的氨基酸序列，采用sgsgs连接102及139位点；第99位点天冬氨酸由半胱氨酸替代，第362位点丝氨酸由半胱氨酸替代，并在替代后的半胱氨酸之间连接内二硫键。其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1-SC1-ID1，其氨基酸序列简称为SEQDB2-CF1-SC1-ID1。牛呼吸道胞合体病毒F蛋白氨基酸序列SEQCAA76980.1第158位亮氨酸由半胱氨酸替代，第290位异亮氨酸由半胱氨酸替代，并由二硫键将两位点连接，同时将第260位点的亮氨酸由半胱氨酸替代，并在该位点实现空穴填充；剔除SEQCAA76980.1序列中103至位点142位点的氨基酸序列，采用sgsgs连接102及143位点；第99位点天冬氨酸由半胱氨酸替代，第362位点丝氨酸由半胱氨酸替代，并在替代后的半胱氨酸之间连接内二硫键。其进行相应的蛋白合成、表达、提纯，得到的牛呼吸道胞合体病毒抗原蛋白简称RD-BRSV-DB2-CF1-SC2-ID1，其氨基酸序列简称为SEQDB2-CF1-SC2-ID1。免疫原性又称抗原性，指抗原刺激机体产生免疫应答能力的特性。免疫反应性指抗原分子能与相应免疫应答的产物（抗体或致敏淋巴细胞），在体内或体外发生特异性结合的性能，采用ELISA法对抗原与抗体的亲和力-免疫进行检验。ELISA法具有灵敏、特异、简单、快速、稳定及易于自动化操作等特点，作为免疫诊断中的一项新技术，已成功地应用于大分子抗原和小分子抗原的定量测定。高亲合力的抗体与抗原的结合力强，即抗原浓度很低时也有较多的抗体结合抗原形成免疫复合物。亲和性用平衡常数K来表示，K值越大，亲和性越高，与抗原结合也越牢固。表1给出了本发明制备的抗原蛋白分别在SiteØ、SiteQP、SiteⅤ、SiteⅡ位点与抗体结合的亲和性数据。表1实施例牛呼吸道胞合体病毒抗原蛋白的特性检测本发明经过设计改造的F-蛋白具有较高的抗体库亲和性。经过改造后的抗原蛋白都保持融合前三聚体刺突状态（Trimer），且都能得到目的抗原蛋白。本发明获得纯化的抗原蛋白与D25抗体进行特异性结合，经蛋白免疫电泳和ELISA方法检测，结果表明不仅具有较好的免疫原性，而且能够诱导小鼠产生较高的抗体水平，表2给出了D25抗体与本发明抗原蛋白特异性结合的免疫原性加权平均值，对照组为非变异野生种。表2实施例牛呼吸道胞合体病毒抗原蛋白与D25特异性结合的特性检测抗原蛋白D25亲和力效价加权平均值（）RD-BRSV-DB14659RD-BRSV-DB2547RD-BRSV-DB1-CF14756RD-BRSV-DB2-CF1875RD-BRSV-DB1-CF1-SC13689RD-BRSV-DB2-CF1-SC14549RD-BRSV-DB1-CF1-SC1-ID110568RD-BRSV-DB1-CF1-SC2-ID11534RD-BRSV-DB2-CF1-SC1-ID11895RD-BRSV-DB2-CF1-SC2-ID15668非变异野生种100对本发明中的抗原蛋白进行物理化学稳定性进行测定，包括温度、pH、渗透压等方面，详细数据见表3，表中数据为在相应条件下的抗原蛋白活性与最佳抗原蛋白活性的比值，结果表明经过设计改造的抗原蛋白对温度、PH值、渗透压等呈现较好的稳定性，具有较高的反应活性。表3实施例牛呼吸道胞合体病毒抗原蛋白的反应活性稳定性实施例2：制备实施例1中RD-BRSV-DB1-CF1-SC1-ID1的具体工艺。看到蛋白机构和形状可以通过以下三种途径，分别为x-射线（x-rayCrystallography）、核磁共振（NMR）、电镜，本发明采用X-射线法对蛋白结构进行观察。采用Solutionmaker600plux系列将目标蛋白进行结晶，将结晶后的目标蛋白采用X-射线系统（SER-CAT22）收集结晶体数据，并使其成像，获得蛋白结构的电子云图，得到关键蛋白F-蛋白侵染前后的数据变化，侵染前后结构变化如图2所示。对上述电子云图进行云数据处理，利用相应的软件（Phenix）和野生蛋白序列进行数据模拟，形成蛋白结构的三维立体结构模拟图形。根据蛋白序列模拟出的三维蛋白结构图形得到F-蛋白的三聚体结构，详见图1。按照晶体化学的原理，利用模拟软件进行抗原蛋白结构设计，其设计包括如图4所示的四种方式：内二硫键（intradisulphidebond）、二硫键（disulphidebond）、空穴填充（cavityfilling）和单链连接（singlechain），对野生种CAA76980.1进行设计。将设计好的蛋白序列转译成DNA序列，采用invitrogen公司提供的Expi293F作为细胞载体在293Fectin培养基中进行培养5天，收集细胞培养液，将目标蛋白进行提纯精制得到设计好的纯化的BRSVF-蛋白，即RD-BRSV-DB1-CF1-SC1-ID1，改造后的牛呼吸道胞合体蛋白抗原侵染牛体后呈现稳定的融合前三聚体刺突状态见图3（左）。上述具体实施方式仅是本发明的具体个案，本发明的专利保护范围包括但不限于上述具体实施方式的产品形态和式样，任何符合本发明权利要求书的一种牛呼吸道胞合体病毒抗原蛋白且任何所属
技术领域：
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