CN116903753B - 一种广谱抗原虫多肽及其制备与应用 - Google Patents
一种广谱抗原虫多肽及其制备与应用 Download PDFInfo
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Abstract
本发明提供了一种广谱抗原虫多肽,其氨基酸序列由转导蛋白结构域序列、linker蛋白序列和突变型APLV4‑VP蛋白序列顺序连接构成;其中,转导蛋白结构域序列为HIV‑TAT转导蛋白结构域序列,linker蛋白序列为柔性linker蛋白序列,突变型APLV4‑VP蛋白序列包括突变型VP4蛋白和原始VP2、VP3、VP1蛋白序列。本发明还提供了一种上述多肽的制备以及在动物体内和/或体外抗原虫的应用。本发明基于突变型APLV4‑VP蛋白,通过转导蛋白结构域实现突变型APLV4‑VP蛋白跨膜转导的可行性,进而设计出一种人工抗原虫多肽;该多肽可用于广谱抗原虫,效果不亚于抗生素类药物且不会导致耐药性。
Description
技术领域
本发明涉及基因重组技术领域及药剂技术领域,尤其涉及一种广谱抗原虫多肽及其制备与应用。
背景技术
重组蛋白是指应用重组DNA或重组RNA技术而获得的蛋白质。重组蛋白工程先应用基因克隆或化学合成技术获得目的基因,连接到适合的表达载体,导入到特定的宿主细胞,利用宿主细胞的遗传系统,表达出有功能的蛋白质分子。
细胞穿膜肽是一类能够穿过细胞膜或组织屏障的短肽。细胞穿膜肽可通过内吞和直接穿透等机制运载蛋白质、RNA、DNA等生物大分子进入细胞内发挥其效应功能。相比于其他非天然的化学分子,细胞穿膜肽具有生物相容性佳、对细胞造成的毒性小、并能与生物活性蛋白直接融合重组表达等优点。许多细胞穿膜肽都是来源于某些直接与宿主细胞相互作用的病毒结构蛋白的蛋白转导结构域。
Apis picorna-like virus 4(APLV4)是一种正链RNA病毒,宿主为蜜蜂和蜂螨,在国内蜂群高发且长期存在。感染该病毒会导致成虫、幼虫和蛹期的显著死亡。除了工蜂、幼虫和蛹的死亡率外,蜂王也会受到直接或间接的影响;在感染APLV4病毒的后期,蜂王体重和产卵量下降。同时该病毒会导致蜂螨的卵、若螨和成螨各阶段的显著死亡。
原虫是一类原生动物,是异养型单细胞真核微生物,基本结构由表膜、细胞质和细胞核三部分组成,大小为1~150μm,结构简单。但其在长期适应寄生生活的进化过程中,发生了细胞质水平的功能分化,产生了一些特化的细胞器,行使高等动物器官样功能。自17世纪列文虎克首次发现兔斯氏艾美耳球虫以来,已记录原虫逾200000种。原虫无处不在,极地的士壤和水体中都可见其踪影。已知原虫中10000余种营寄生生活,一些原虫是重要病原,可引起动物和人类的一些重要疾病,如疟疾、球虫病、梨形虫病、毛滴虫病等,给人类健康和经济发展造成了巨大伤害。目前的治疗手段主要是硝唑类抗生素。随着禁抗令的实施,抗生素的使用越来越值得关注,且抗生素的使用会导致原虫耐药性的出现。对于养殖企业来说临出栏产品出现原虫病会是非常严重的打击。据此,一种安全的有效的非抗生素类的抗原虫药物,成为广大养殖企业的迫切需求。
目前,专利CN101048154A公开了一种抗原虫剂,但该药物只针对利士曼原虫,非广谱抗原虫药物,不适合混合感染的条件下使用,而国内养殖的突出问题就是混合感染。因此,CN101048154A公布的方法仍无法满足现阶段国内养殖行业的使用需求。
发明内容
本发明所要解决的技术问题在于提供一种广谱抗原虫多肽及其制备与应用,其基于突变型APLV4-VP蛋白以及转导蛋白结构域(作为细胞穿膜肽)进行设计,设计出一种人工的抗原虫多肽,该多肽可用于广谱抗原虫,效果不亚于抗生素类药物且不会导致耐药性。
本发明采用以下技术方案解决上述技术问题:
一种广谱抗原虫多肽,所述广谱抗原虫多肽的氨基酸序列由转导蛋白结构域序列、linker蛋白序列和突变型APLV4-VP蛋白序列顺序连接构成;其中,所述转导蛋白结构域序列为HIV-TAT转导蛋白结构域氨基酸序列;所述linker蛋白序列为柔性linker蛋白氨基酸序列(GGGGS)n,n≥1;所述突变型APLV4-VP蛋白序列包括顺序连接的突变型VP4蛋白氨基酸序列和原始VP2、VP3、VP1蛋白氨基酸序列。
作为本发明的优选方式之一,所述HIV-TAT转导蛋白结构域氨基酸序列如SEQ IDNO.1所示;所述柔性linker蛋白氨基酸序列如SEQ ID NO.2所示;所述突变型APLV4-VP蛋白中,突变型VP4蛋白氨基酸序列如SEQ ID NO.3所示,未突变的原始VP2、VP3、VP1蛋白氨基酸序列分别如SEQ ID NO.4、5、6所示,完整的突变型APLV4-VP蛋白氨基酸序列如SEQ ID NO.7所示。
一种上述广谱抗原虫多肽的制备方法,包括如下步骤:
(1)广谱抗原虫多肽基因序列的获得;
(2)工程菌构建;
(3)发酵纯化,获得广谱抗原虫多肽。
作为本发明的优选方式之一,所述步骤(1)中广谱抗原虫多肽基因序列的获得,具体获取方法为:
①获取转导蛋白结构域序列;
②获取突变型APLV4-VP蛋白序列;
③使用linker蛋白序列将所述转导蛋白结构域序列和所述突变型APLV4-VP蛋白序列进行连接,得到广谱抗原虫多肽的氨基酸序列;
④使用DNAMAN软件,做反向翻译,结合Bacillus subtilis密码子偏好进行优化,并于上游添加酶切位点BamHⅠ,下游添加酶切位点XbaⅠ,得广谱抗原虫多肽的基因序列。
作为本发明的优选方式之一,所述步骤(2)中工程菌构建,具体构建方法为:通过内切酶对获得的广谱抗原虫多肽的基因序列及pHT43质粒进行双酶切,将双酶切产物使用T4连接酶连接,挑取阳性单菌落、恒温扩大培养、送检测序合格后获得。
作为本发明的优选方式之一,所述步骤(3)中发酵纯化,具体发酵纯化方法为:
①取经测序合格的工程菌于摇床37℃、220rpm培养至OD600为2.0;加入3mM的IPTG,随即将摇床的温度调为18℃,诱导12h后收获;于4℃,12000rpm离心15min,取上清弃沉淀;
②取离心后上清液1:1加入80%饱和度的(NH4)2SO4;然后将该混合液放于4℃冰箱静置48h,将发酵液中的蛋白絮凝沉淀析出;于4℃、12000rpm离心15min,取沉淀,弃去上清液;用PBS缓冲液对沉淀进行悬浮,并用与悬浮液相同浓度的PBS透析去除盐分;取透析后的蛋白溶液1:1加入80%饱和度的(NH4)2SO4;然后将该混合液放于4℃冰箱静置48h,将混合液中的蛋白絮凝沉淀析出;于4℃,12000rpm离心15min,取沉淀,弃去上清液;用PBS缓冲液对沉淀进行悬浮,并用与悬浮液相同浓度的PBS透析去除盐分即得目的蛋白。
一种上述广谱抗原虫多肽在动物体内和/或体外抗原虫的应用。
作为本发明的优选方式之一,当动物口服广谱抗原虫多肽后,所述广谱抗原虫多肽在动物体内发挥广谱抗原虫作用。
作为本发明的优选方式之一,当在动物饲养环境中喷洒广谱抗原虫多肽后,所述广谱抗原虫多肽在动物体外发挥广谱抗原虫作用。
本发明相比现有技术的优点在于:
(1)本发明基于突变型APLV4-VP蛋白进行设计,通过转导蛋白结构域前置来实现突变型APLV4-VP蛋白跨膜转导的可行性,进而设计出一种人工的抗原虫多肽(转导蛋白结构域作为细胞穿膜肽,引导APLV4-VP蛋白进入细胞,提高抗原虫效果);该多肽可用于广谱抗原虫,效果不亚于抗生素类药物且不会导致耐药性;
(2)本发明采用枯草芽孢杆菌发酵表达,产物的后处理工艺简单,成本低廉,可广泛适用于各类养殖业使用。
附图说明
图1是实施例2中蜜蜂组织研磨液于Vero细胞连续增殖17代后CPE图(图1中,A图为蜜蜂组织的CPE,B图为Vero细胞对照);
图2是实施例2中突变型APLV4-VP蛋白测序结果与未突变VP序列比对结果图;
图3是实施例3中广谱抗原虫多肽的Page电泳图(图3中,M:蛋白Marker26610;泳道1:重组广谱抗原虫多肽;泳道2:空菌对照);
图4是实施例4中RTCA SP Instrument系统导出的细胞生长全过程图(图4中,纵坐标代表细胞生长指数CGI,横坐标代表培养时间)。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
下述实施例中的实验方法,如无特殊说明,为常规方法。下述实施例中所用的试验材料,如无特殊说明,为现有生化试剂商店直接购买得到的常规材料。
实施例1:广谱抗原虫多肽
所述广谱抗原虫多肽的氨基酸序列由转导蛋白结构域序列、linker蛋白序列和突变型APLV4-VP蛋白序列顺序连接构成。
所述转导蛋白结构域序列为HIV-TAT转导蛋白结构域氨基酸序列,如SEQ ID NO.1所示。
所述linker蛋白序列为柔性linker蛋白氨基酸序列(GGGGS)n,n≥1,如SEQ IDNO.2所示。
所述突变型APLV4-VP蛋白序列包括顺序连接的突变型VP4蛋白氨基酸序列和未突变的原始VP2、VP3、VP1蛋白氨基酸序列。其中,突变型VP4蛋白氨基酸序列如SEQ ID NO.3所示,未突变的原始VP2、VP3、VP1蛋白氨基酸序列分别如SEQ ID NO.4、5、6所示,完整的突变型APLV4-VP蛋白氨基酸序列如SEQ ID NO.7所示。
实施例2:突变型APLV4-VP蛋白序列的获得
我司在研发梅氏热厉螨(小蜂螨)治疗药物过程中发现试验蜂场内一蜂群经镜检未有孢子虫感染,而试验蜂场内其他蜂群经检查均有孢子虫感染。经实验排除抗小蜂螨药物作用。经16S rRNA RT-PCR扩增及测序排除致病菌感染。
取该蜂群蜜蜂数只组织研磨,接种于Vero细胞中,使用含150ug/mL胰酶的无血清培养基连续增殖17代后细胞病变CPE明显(如图1所示,CPE呈合胞体并破碎状),反复冻融三次使用离心机12000rpm离心去除细胞碎片,后使用蔗糖密度梯度离心法纯化。采用RT-PCR及PCR方法检测,排除残翅病毒、西奈湖病毒等常见的12种病毒感染。将纯化后的样品送华大基因及生工生物分别做全基因测序及N端测序。经BLAST发现,与现有Apis picorna-likevirus 4高度同源但在VP 4有部分突变(序列比对结果如图2)。取结构蛋白氨基酸序列:VP4(突变)-VP2-VP3-VP1如SEQ ID NO.7所示,记为突变型APLV4-VP蛋白。
实施例3:广谱抗原虫多肽的制备
(1)TAT-VP融合蛋白基因序列的获得
参考NCBI登录号8CCZ_C,取HIV-TAT序列,得HIV-TAT转导蛋白结构域氨基酸序列,如SEQ ID NO.1所示。
依照实施例2中方法获得突变型APLV4-VP蛋白的氨基酸序列,如SEQ ID NO.7所示。
使用如SEQ ID NO.2所示的linker蛋白将HIV-TAT转导蛋白结构域序列和突变型APLV4-VP蛋白的氨基酸序列进行连接,得到目的蛋白氨基酸序列,如SEQ ID NO.8所示。
使用DNAMAN软件,做反向翻译,结合Bacillus subtilis密码子偏好做优化,并于上游添加酶切位点BamHⅠ和起始密码子,下游添加终止密码子和酶切位点XbaⅠ,得广谱抗原虫多肽的人工核酸序列,如SEQ ID NO.9所示;基因序列送华大基因合成,测序合格,获得目的蛋白的核酸序列。
(2)工程菌构建
对上述合成的目的基因,使用BamHⅠ和XbaⅠ两种内切酶对合成的基因及pHT43质粒进行双酶切;将双酶切产物使用连接酶连接,并导入WB800N感受态细胞中;涂布于氨苄LB平板上,37℃恒温培养;挑取氨苄LB平板上长出的单菌落扩大培养并取样送华大基因测序;测序结果与目的基因一致,表示表达菌构建成功,记为TAT-VP工程菌。
(3)发酵纯化
取经测序合格的TAT-VP工程菌使用LB培养基于摇床37℃、220rpm培养至OD600为2.0;加入终浓度3mM的IPTG,随即将摇床的温度调为18℃,诱导约12h后收获;于4℃,12000rpm离心15min,取上清弃沉淀;取离心后上清液1:1加入80%饱和度的(NH4)2SO4;然后将该混合液放于4℃冰箱静置48h,将发酵液中的蛋白絮凝沉淀析出;于4℃,12000rpm离心15min,取沉淀,弃去上清液;用PBS缓冲液对沉淀进行悬浮,并用与悬浮液相同浓度的PBS透析去除盐分;取透析后的蛋白溶液1:1加入80%饱和度的(NH4)2SO4;然后将该混合液放于4℃冰箱静置48h,将混合液中的蛋白絮凝沉淀析出;于4℃,12000rpm离心15min,取沉淀,弃去上清液;用PBS缓冲液对沉淀进行悬浮,并用与悬浮液相同浓度的PBS透析去除盐分。
做SDS-Page初步鉴定,目的蛋白大小与预期一致(18kD,如图3所示)。所得蛋白即为目的蛋白—广谱抗原虫多肽,记为Anti-protozoon polypeptide,APP。
实施例4:广谱抗原虫多肽体外抗原虫效果验证
(1)参数设置
RTCA SP Instrument系统上新建实验,设定:
Step 1:1;1min;基线测量,固定检测1min 1次;
Step 2:36;30min;18hrs,用于检测细胞增殖过程;
Step 3:144;30min;72hrs,用于检测原虫攻虫及药物作用;
(2)细胞培养
Vero细胞以1.5×104/孔接种于安捷伦RTCA E-Plate 96孔细胞板上,5%CO2培养箱中于RTCA SP Instrument系统上培养24h后进行攻虫实验。
(3)攻虫及评价
在E-Plate 96孔细胞板上以100个/孔攻艾美尔球虫32孔,以100个/孔攻疟原虫16孔,以100个/孔攻毛滴虫16孔。剩余32孔不攻任何原虫,加入稀释液(2%CS的DMEM)。
与此同时,攻艾美尔球虫的组别中,8孔加入2ug的TAT-Linker多肽(HIV-TAT转导蛋白结构域+柔性linker蛋白),8孔加入2ug的VP多肽(突变型APLV4-VP蛋白),8孔加入2ug的本发明目的蛋白(HIV-TAT转导蛋白结构域+柔性linker蛋白+突变型APLV4-VP蛋白,APP),8孔加入稀释液。攻疟原虫的组别中,8孔加入2ug的本发明目的蛋白(APP),8孔加入稀释液。攻毛滴虫的组别中,8孔加入2ug的本发明目的蛋白(APP),8孔加入稀释液。不攻原虫的组别中,8孔加入20ug的TAT-Linker多肽,8孔加入2ug的VP多肽,8孔加入2ug的APP,8孔加入稀释液。5%CO2培养箱中于RTCA SP Instrument系统上培养72h。
导出细胞在RTCA SP Instrument系统上的生长曲线。如图4所示,其中纵坐标代表细胞生长指数(Cell growth index,CGI)是细胞活力和细胞数量的拟合值,横坐标代表培养时间。
由图4可知:使用本发明目的蛋白(广谱抗原虫多肽,APP)处理的攻虫组较空白对照组及阴性对照组CGI略有降低,但较三组攻虫组都有较好的生长增殖活性。且在球虫攻虫组内,明显可以看到只有柔性linker连接TAT的VP组(即本发明目的蛋白,APP)有较好的效果,单纯的VP无效,由此也可见VP是通过TAT转导进入胞内(细胞和原虫细胞)发挥抗原虫作用。
实施例5:广谱抗原虫多肽体内抗原虫效果验证
(1)试验动物培育
试验动物选择艾美尔球虫、疟原虫及毛滴虫都敏感的鸡。选用SPF鸡胚于无菌环境中孵化,并于无艾美尔球虫、疟原虫及毛滴虫的环境中饲养,自由采食和饮水。
(2)药物处理
广谱抗原虫多肽冻干后,试验剂量为每吨饲料有效添加量为100克;抗球虫对照药物选用二硝托胺,试验剂量为每吨饲料添加量为125克;抗疟原虫对照药物选用磷酸氯喹,试验剂量为每吨饲料添加量为250克;抗毛滴虫对照药物选用二甲硝咪唑,试验剂量为每吨饲料添加量为400克。
(3)攻虫与治疗分组
艾美尔球虫、疟原虫及毛滴虫均培育至孢子化,保存备用。
SPF小鸡饲喂至14天龄,剔除体重过大、过小的鸡只,称重,记录初重。随机分组,每组10只。按下表1进行分组。
表1攻虫及治疗分组
15天龄时,所有需要攻虫的组,每只鸡口服给予相应原虫1万个虫卵。每日观察并记录各组鸡饮水、采食、粪便以及死亡等情况,及时剖解死亡鸡,分析死亡原因。攻虫7天后,记录各组的存活率,途中死亡鸡剖检,分析死亡原因。存活鸡脱颈处死。称重。取盲肠,刮取盲肠粘膜和内容物,检测计算每只鸡卵囊数。
(4)抗原虫效果评价
设定抗原虫指数(Antiprotozoan Index,API),API=(存活率+相对增重率)-(病变值+卵囊值);存活率=(试验结束时存活鸡只数/试验鸡只数)×100%;相对增重率=(试验组增重/空白对照组增重)×100%;API≥160判为优秀,API=130~159判为良好,API=100~129为低敏,API<100判为无效。
设定病变记分减少率(Reduction of lesion scores,RLS),RLS=(感染不用药对照组平均病变记分-感染用药组平均病变记分)/感染不用药对照组平均病变记分×100%。RLS≥50%为完全敏感。
试验各组抗原虫指数(API)和RLS结果见表2。
表2试验各组抗原虫指数(API)及病变记分减少率(RLS)
本次试验每只鸡攻虫剂量属较低攻虫剂量。从抗原虫指数(API)来看,广谱抗原虫多肽API值对不同原虫的值169.1、165.5、159.9、149.8都较好,属于良好以上,相比三种化药分别要高21%、34%、20%、60%。广谱抗原虫多肽较化药从RLS来看都高度敏感。广谱抗原虫多肽较化药从卵囊值看普遍效果要好。广谱抗原虫多肽较化药从增重效果看都较好。综合来说,在体内抗原虫效果上,广谱抗原虫多肽要优于化学抗生素药物。
据此,本发明广谱抗原虫多肽可在动物体内和/或体外抗原虫上进行应用。当动物口服广谱抗原虫多肽,所述广谱抗原虫多肽可在动物体内发挥广谱抗原虫作用。当在动物饲养环境中施放广谱抗原虫多肽,所述广谱抗原虫多肽可在动物体外发挥广谱抗原虫作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (4)
1. 一种广谱抗原虫多肽,其特征在于,所述广谱抗原虫多肽的氨基酸序列由转导蛋白结构域序列、linker蛋白序列和突变型APLV4-VP蛋白序列顺序连接构成;其中,所述转导蛋白结构域序列为HIV-TAT转导蛋白结构域氨基酸序列,如SEQ ID NO.1所示;所述linker蛋白序列为柔性linker蛋白氨基酸序列(GGGGS)n,n≥1,如SEQ ID NO.2所示;所述突变型APLV4-VP蛋白序列包括顺序连接的突变型VP4蛋白氨基酸序列和原始VP2、VP3、VP1蛋白氨基酸序列,完整氨基酸序列如SEQ ID NO.7所示。
2.一种如权利要求1所述的广谱抗原虫多肽的制备方法,其特征在于,包括如下步骤:
(1)广谱抗原虫多肽基因序列的获得;
(2)以枯草芽孢杆菌为表达宿主菌株,构建工程菌;
(3)发酵纯化,获得广谱抗原虫多肽;具体发酵纯化方法为:
①取经测序合格的工程菌于摇床37℃、220rpm培养至OD600为2.0;加入3mM的IPTG,随即将摇床的温度调为18℃,诱导12h后收获;于4℃,12000rpm离心15min,取上清弃沉淀;
②取离心后上清液1:1加入80%饱和度的(NH4)2SO4;然后将该混合液放于4℃冰箱静置48h,将发酵液中的蛋白絮凝沉淀析出;于4℃、12000rpm离心15min,取沉淀,弃去上清液;用PBS缓冲液对沉淀进行悬浮,并用与悬浮液相同浓度的PBS透析去除盐分;取透析后的蛋白溶液1:1加入80%饱和度的(NH4)2SO4;然后将该混合液放于4℃冰箱静置48h,将混合液中的蛋白絮凝沉淀析出;于4℃,12000rpm离心15min,取沉淀,弃去上清液;用PBS缓冲液对沉淀进行悬浮,并用与悬浮液相同浓度的PBS透析去除盐分即得目的蛋白。
3.根据权利要求2所述的广谱抗原虫多肽的制备方法,其特征在于,所述步骤(1)中广谱抗原虫多肽基因序列的获得,具体获取方法为:
①获取转导蛋白结构域序列;
②获取突变型APLV4-VP蛋白序列;
③使用linker蛋白序列将所述转导蛋白结构域序列和所述突变型APLV4-VP蛋白序列进行连接,得到广谱抗原虫多肽的氨基酸序列;
④使用DNAMAN软件,做反向翻译,结合Bacillus subtilis密码子偏好进行优化,并于上游添加酶切位点BamHⅠ,下游添加酶切位点XbaⅠ,得广谱抗原虫多肽的基因序列。
4.根据权利要求2所述的广谱抗原虫多肽的制备方法,其特征在于,所述步骤(2)中工程菌构建,具体构建方法为:通过内切酶对获得的广谱抗原虫多肽的基因序列及pHT43质粒进行双酶切,将双酶切产物使用T4连接酶连接,挑取阳性单菌落、恒温扩大培养、送检测序合格后获得。
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