CN114853913A - 一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用 - Google Patents
一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用 Download PDFInfo
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- CN114853913A CN114853913A CN202210721011.3A CN202210721011A CN114853913A CN 114853913 A CN114853913 A CN 114853913A CN 202210721011 A CN202210721011 A CN 202210721011A CN 114853913 A CN114853913 A CN 114853913A
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Abstract
本发明公开了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用,包括所述融合蛋白的核苷酸序列如SEQ ID NO:1所示,所述融合蛋白的氨基酸序列如SEQ ID NO:2所示,本发明还提供了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体及构建方法,所述表达载体包含所述融合蛋白的核苷酸。本发明属于基因工程技术领域;本发明通过融合植物抗菌肽AFP1与分泌信号肽SPamyQ设计得到融合蛋白AFP1‑S,有效地保障了抗菌肽AFP1在肠黏膜层释放,抑杀肠道内病原微生物,调节免疫应答,进一步本发明通过体外抑菌拮抗试验证实了融合蛋白对大肠杆菌、金黄色葡萄球菌、沙门氏菌、铜绿假单胞菌和白色念珠菌的抑菌活性,本发明可应用于预防和治疗禽畜细菌性腹泻。
Description
技术领域
本发明属于基因工程技术领域,具体是指一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用。
背景技术
大肠杆菌、金黄色葡萄球菌、沙门氏菌、铜绿假单胞菌和白色念珠菌等各种细菌以及动物寄生虫、真菌、病毒等导致的感染长期以来是畜牧、禽蛋养殖中瓶颈限制问题,严重阻碍畜牧业的发展,尤其每年由细菌性感染导致的直接经济损失高达数十亿元,由于抗生素的长期使用产生耐药菌,耐药菌引起的腹泻与感染日益严重,自抗生素禁用以后,畜牧业中主要以添加中药和益生菌解决临床疾病预防问题,但中药材成本高,长期使用后在动物体内残留、富集,直接影响了畜产品的品质和间接损害人类的健康,而普通益生菌功效不足,因此亟待需要安全、新型抗生素替代品来控制疾病和促进动物健康生长。
芽孢杆菌、酵母菌和乳酸菌是应用最多的微生物添加剂菌种,芽孢杆菌由于稳定性好、抗逆性强、复活率高,通过与病原菌竞争营养物质、抑制病原菌等提高机体的免疫机能,并提供营养物质等作用,调节消化道健康,增强动物体的免疫功能,达到促进目标动物的生长、提高饲料的转化率的目的。
植物防御素抗菌肽(英文简写为AFP1)仅由51个氨基酸组成,是一种小而富含半胱氨酸的肽,核磁共振分析显示,AFP1由一个α-螺旋和一个三链反平行β片组成,研究发现,AFP1对人类HepG2细胞无毒性,最高可达40 μM,因此AFP1缺乏一般的细胞毒性活性,目前,在畜牧业中尚未被应用于防治由细菌性感染造成的腹泻等疾病,此外,由于抗菌肽AFP1来源于植物(真核生物)中,因此直接在原核生物中表达受限,表达量低下,以及易被降解、无法进入禽畜的肠道内进行抑菌杀菌作用。
发明内容
针对上述情况,为克服现有技术的缺陷,本发明提供了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用,为了解决植物来源抗菌肽AFP1在原核生物中表达量低下、易被降解的问题,本发明通过融合植物抗菌肽AFP1与分泌信号肽SPamyQ设计得到融合蛋白AFP1-S,有效地保障了抗菌肽AFP1在肠黏膜层释放,抑杀肠道内病原微生物,调节免疫应答,进一步本发明通过体外抑菌拮抗试验证实了本发明提供的融合蛋白对大肠杆菌、金黄色葡萄球菌、沙门氏菌、铜绿假单胞菌和白色念珠菌的抑菌活性,本发明首次报道应用植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白治疗细菌性感染导致的禽畜类疾病,降低了抗生素的使用风险并提高了经济效益。
为了实现上述目的,本发明采取的技术方案如下:本发明提出了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白,所述融合蛋白的核苷酸序列如SEQ ID NO:1所示,所述融合蛋白的氨基酸序列如SEQ ID NO:2所示。
优选地,所述融合蛋白的表达宿主为大肠杆菌、毕赤酵母、枯草芽孢杆菌中的一种。
进一步,本发明还提供了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体,所述表达载体包含所述融合蛋白的核苷酸。
进一步地,本发明还提供了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体的构建方法,包括如下步骤:
步骤一:以人工合成的基因AFP1-S为模板,使用引物进行PCR扩增得到带有酶切位点的AFP1-S片段;
步骤二:使用限制性内切酶切割载体与AFP1-S片段,采用同源重组酶连接载体与AFP1-S片段得到连接产物;
步骤三:将所述连接产物转化大肠杆菌感受态中获得阳性克隆子,然后提取所述阳性克隆子的质粒,经过DNA测序验证后,得到所述融合蛋白的表达载体。
优选地,所述载体为pWUKANG01,所述融合蛋白的表达载体为pWUKANG01-AFP1-S,所述限制性内切酶为XholⅠ。
优选地,所述引物为5’-GCGGTACCGAGCTCGCTCGAG-3’和5’-TGCAGCGGCTAGCCCCTCGAGTCA-3’,所述引物与目的基因互补,并且提供限制性内切酶的保护信号,所述引物上设有与载体上15-18 bp的同源序列,可以更方便的与载体连接。
本发明还提供了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的应用,包括如下任一一条:
(1)所述融合蛋白应用于构建预防和治疗禽畜细菌性腹泻的工程菌株;
(2)所述融合蛋白应用于制备预防和治疗禽畜细菌性腹泻的药物。
优选地,所述细菌性腹泻包括由沙门氏菌、大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌和白色念珠菌的一种或者多种引起的腹泻。
采用上述方案本发明取得的有益效果如下:本方案提出了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用:
(1)本发明通过设置芽孢杆菌内源的分泌肽SPamyQ片段保护了植物抗菌肽AFP1的独立折叠,使融合蛋白分泌后在枯草芽孢杆菌外周被分泌蛋白酶激酶识别并切割成独立的活性结构域,有效地保障了抗菌肽AFP1在肠黏膜层释放,抑杀肠道内病原微生物,调节免疫应答;
(2)分泌性表达载体pWUKANG01在多克隆位点前面没有分泌信号肽序列,因此设计补充分泌信号肽SPamyQ与植物抗菌肽AFP1融合,分泌信号肽能高效的将重组蛋白分泌到胞外;
(3)表达载体pWUKANG01-AFP1-S允许在细胞质中高水平表达重组蛋白;
(4)表达载体pWUKANG01-AFP1-S能够去除在克隆过程中所使用的Amp抗性基因片段,通过ECOR Ⅰ,可实现双酶切去除Amp基因,从而避免抗性基因的影响;
(5)本发明中所述融合蛋白对大肠杆菌、金黄色葡萄球菌、沙门氏菌、铜绿假单胞菌和白色念珠菌均具有显著抑制作用,枯草芽孢杆菌BS168-AFP1-S分泌表达的融合蛋白复合物可以通过直接的抑杀大肠杆菌、金黄色葡萄球菌、沙门氏菌、铜绿假单胞菌和白色念珠菌等修复受损的肠道黏膜;
(6)所述融合蛋白在分泌表达后在芽孢杆菌体外和肠道内自动分割为单独的活性蛋白,融合蛋白能够调节各类肠道细胞的细胞周期,加速修复与提高肠道免疫力,肠道黏膜层富集有大量的粒细胞与巨噬细胞,而肠系膜淋巴结富集有大量的免疫淋巴细胞,融合蛋白在肠道黏膜和肠系膜淋巴结同时行使功能并发挥作用,既可以杀灭感染的细菌,也可以同时修复肠道,提高免疫力,实现多种益生功能;
(7)本发明中所述融合蛋白应用广泛,可以降低腹泻率,提高日增重及饲料消化率,降低料肉比,提高蛋壳厚度及质量等。
附图说明
图1为使用PCR扩增重组基因AFP1-S片段的结果图;
图2为表达载体pWUKANG01-AFP1-S质粒电泳凝胶图;
图3为枯草芽孢杆菌BS168-AFP1-S对大肠杆菌的抑制效果;
图4为枯草芽孢杆菌BS168-AFP1-S对沙门氏菌的抑制效果;
图5为枯草芽孢杆菌BS168-AFP1-S对金黄色葡萄球菌的抑制效果;
图6为枯草芽孢杆菌BS168-AFP1-S对白色念珠球菌的抑制效果;
图7为枯草芽孢杆菌BS168-AFP1-S对铜绿假单胞菌的抑制效果。
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例;基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法;下述实施例中所用的试验材料,如无特殊说明,均为从商业渠道购买得到的。
本发明提供了一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用,所述融合蛋白的核苷酸序列如SEQ ID NO:1所示,所述融合蛋白的氨基酸序列如SEQ ID NO:2所示。
值得注意的是,由于遗传密码子兼并性原则,因此本发明的融合蛋白序列还可以由其他核酸密码子组合翻译出来,因此其他可以编码为本发明中的所述融合蛋白的氨基酸序列的核苷酸均属于本发明保护的范围。
实施例1
一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体的构建
由于抗菌肽AFP1来源于植物(真核生物)中,因此直接在原核生物中表达受限,为了实现抗菌肽AFP1的高效表达,本发明通过融合抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ设计得到融合蛋白的核苷酸序列SEQ ID NO:1所示,融合蛋白的氨基酸序列SEQ ID NO:2所示,其中SPamyQ片段的氨基酸序列为MIQKRKRTDSFVQTCAYVHAVIVSLPITKTSA,植物抗菌肽AFP1的氨基酸序列为DGVKLCDVPSGTWSGHCGSSSKCSQQCKDREHFA,再根据所述融合蛋白的核苷酸序列,使用DNA人工合成技术得到基因AFP1-S。
(1)以人工合成的基因AFP1-S为模板,使用引物进行PCR扩增得到带有酶切位点的AFP1-S片段,其中PCR扩增的引物为5’-GCGGTACCGAGCTCGCTCGAG-3’和5’-TGCAGCGGCTAGCCCCTCGAGTCA-3’;
PCR扩增条件为:94℃ 1 min;94℃ 10 s,52℃ 10 s,68℃ 10 s,5个循环;94℃10 s, 68℃ 15 s,30个循环;68℃ 1 min;
(2)按照说明书使用限制性内切酶xhol I(购自Fermentals-上海玉博生物科技有限公司)双酶切载体pWUKANG01(购于德国MoBi TEC中国北京代理公司)与AFP1-S片段,然后将AFP1-S片段使用同源重组连接酶(购自生工生物工程上海股份有限公司)连接到pWUKANG01质粒上得到连接产物;
(3)将连接产物转化入大肠杆菌感受态获得阳性克隆子,提取阳性克隆子的质粒,使用DNA测序并验证后,得到所述融合蛋白的表达载体pWUKANG01-AFP1-S。
如图1所示,通过PCR扩增得到重组基因AFP1-S;如图2所示,通过上述操作获得所述融合蛋白的表达载体pWUKANG01-AFP1-S,图中1表示所述融合蛋白的表达载体pWUKANG01-AFP1-S,2表示进行双酶切后的载体pWUKANG01。
实施例2
一种表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的宿主菌株的构建
将实施例1中获得的所述融合蛋白的表达载体pWUKANG01-AFP1-S使用电击法转化入枯草芽孢杆菌BS168,枯草芽孢杆菌BS168购自即四川省工业微生物菌种保藏管理中心。
具体试验步骤为:
(1)使用灭菌后的接种针挑取枯草芽孢杆菌BS168的单一菌落于5 mL LB液体培养基中,与37℃,180 rpm过夜培养;
(2)吸取实施例2的步骤(1)中培养的枯草芽孢杆菌BS168于50 mL GM培养基中(LB液体培养基中加入0.5 mol/L 山梨醇),37℃,200 rpm培养3-4 h至OD600=1.0即可得到枯草芽孢杆菌BS168预处理菌液;
(3)取枯草芽孢杆菌BS168预处理菌液冰浴10 min,然后使用5000 rpm,8min,4℃离心收集菌体;
(4)用40 mL预冷的电转缓冲液ETM(0.5 mol/L 山梨醇,0.5 mol/L 甘露醇,10%甘油)洗涤菌体,5000 rpm,8 min,4℃离心去上清,重复操作3次;
(5)将洗涤后的枯草芽孢杆菌BS168菌体重悬于500 μL的电转缓冲液ETM中,每管60 μL进行分装,即可得到枯草芽孢杆菌BS168感受态细胞;
(6)将60 μL枯草芽孢杆菌BS168感受态细胞中加入5 μL载体质粒pWUKANG01-AFP1-S,冰浴5 min,加入预冷的电转杯中,电击一次,其中电转仪设置:2.0 kv,25 μF,200Ω,电击1次,持续时间4.5-5 ms;
(7)电击完毕立即加入1 mL 复苏培养基RM(LB液体培养基中加入0.5 mol/L山梨醇和0.38 mol/L甘露醇),37℃,200 rpm,培养3 h后,将枯草芽孢杆菌BS168感受态细胞涂布于LB固体培养基,于37℃过夜培养,再次使用DNA测序并验证后即可得到表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的宿主菌株枯草芽孢杆菌BS168-AFP1-S。
实施例3
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白对大肠杆菌抑菌效果分析
大肠杆菌生活在多种动物的肠道中,一些致病性的大肠杆菌是引起禽畜发生腹泻的主要原因,因此可以通过测量由实施例2中制备的枯草芽孢杆菌BS168-AFP1-S对大肠杆菌的抑制效果验证所述融合蛋白的功能,本发明使用的菌株为大肠杆菌标准菌株ATCC25922(购于美国ATCC细胞库),具体操作步骤如下:
(1)将购买的大肠杆菌菌种接种到LB液体培养基中,37℃,200 rpm,培养12 h,使用LB液体培养基稀释培养得到的大肠杆菌菌液至OD600值为0.8得到大肠杆菌实验菌液;
(2)将大肠杆菌实验菌液接种于96孔板中,每个孔槽接种200 μL;
(3)将枯草芽孢杆菌BS168-AFP1-S接种于孔槽中,每个空槽接种10μL;
(4)37℃静置培养,于18 h测量OD600值;
设置的处理为:AFP1原液+大肠杆菌、AFP1 LB稀释2倍+大肠杆菌、AFP1 LB稀释4倍+大肠杆菌、AFP1 LB稀释8倍+大肠杆菌、AFP1 LB稀释16倍+大肠杆菌,以LB+大肠杆菌为空白对照,单个处理设置7个重复,实验重复3次。
抑菌效果判定标准:以阴性对照孔OD600值为参照,高于此值的孔槽为促进抑菌生长,低于此值的孔槽为抑菌细菌生长,具有抑菌活性。
如图3所示,表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的枯草芽孢杆菌BS168-AFP1-S分泌的融合蛋白溶液可以抑制大肠杆菌,并且稀释2倍和稀释16倍后仍然具备较高的抑制效果。
实施例4
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白对沙门氏菌抑菌效果分析
沙门氏菌属于无宿主特异性而有侵害性的病原菌之一,因此可以通过测量由实施例2中制备的枯草芽孢杆菌BS168-AFP1-S对沙门氏菌的抑制效果验证所述融合蛋白的功能,本发明使用菌株为沙门氏菌标准菌株ATCC58785(购于美国ATCC细胞库),具体操作步骤如下:
(1)将购买的沙门氏菌菌种接种到LB液体培养基中,37℃,200 rpm,培养12 h,使用LB培养基稀释培养得到的沙门氏菌菌液至OD600值为0.8得到沙门氏菌实验菌液;
(2)将沙门氏菌实验菌液接种于96孔板中,每个孔槽接种200 μL;
(3)将枯草芽孢杆菌BS168-AFP1-S接种于孔槽中,每个空槽接种10μL;
(4)37℃静置培养,于18 h测量OD600值;
设置的处理为:AFP1原液+沙门氏菌、AFP1 LB稀释2倍+沙门氏菌、AFP1 LB稀释4倍+沙门氏菌、AFP1 LB稀释8倍+沙门氏菌、AFP1 LB稀释16倍+沙门氏菌,以LB+沙门氏菌为空白对照,单个处理设置7个重复,实验重复3次。
抑菌效果判定标准:以阴性对照孔OD600值为参照,高于此值的孔槽为促进抑菌生长,低于此值的孔槽为抑菌细菌生长,具有抑菌活性。
如图4所示,表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的枯草芽孢杆菌BS168-AFP1-S分泌的融合蛋白溶液可以抑制沙门氏菌,并且稀释2倍和稀释8倍后仍然具备较高的抑制效果。
实施例5
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白对金黄色葡萄球菌抑菌效果分析
金黄色葡萄球菌隶属于葡萄球菌属,是革兰氏阳性菌代表,为一种常见的食源性致病微生物,因此可以通过测量由实施例2中制备的枯草芽孢杆菌BS168-AFP1-S对金黄色葡萄球菌的抑制效果验证所述融合蛋白的功能,本发明使用的菌株为金黄色葡萄球菌标准菌株ATCC25922(购于美国ATCC细胞库),具体操作步骤如下:
(1)将购买的金黄色葡萄球菌菌种接种到LB液体培养基中,37℃,200 rpm,培养12h,使用LB培养基稀释培养得到的金黄色葡萄球菌菌液至OD600值为0.8得到金黄色葡萄球菌实验菌液;
(2)将金黄色葡萄球菌实验菌液接种于96孔板中,每个孔槽接种200 μL;
(3)将枯草芽孢杆菌BS168-AFP1-S接种于孔槽中,每个空槽接种10μL;
(4)37℃静置培养,于18 h测量OD600值;
设置的处理为:AFP1原液+金黄色葡萄球菌、AFP1 LB稀释2倍+金黄色葡萄球菌、AFP1 LB稀释4倍+金黄色葡萄球菌、AFP1 LB稀释8倍+金黄色葡萄球菌、AFP1 LB稀释16倍+金黄色葡萄球菌,以LB+金黄色葡萄球菌为空白对照,单个处理设置7个重复,实验重复3次。
抑菌效果判定标准:以阴性对照孔OD600值为参照,高于此值的孔槽为促进抑菌生长,低于此值的孔槽为抑菌细菌生长,具有抑菌活性。
如图5所示,表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的枯草芽孢杆菌BS168-AFP1-S分泌的融合蛋白溶液可以抑制金黄色葡萄球菌,并且稀释2倍和稀释16倍后仍然具备较高的抑制效果。
实施例6
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白对白色念珠菌抑菌效果分析
白色念珠菌多生存在动物肠道中,在禽畜免疫力下降或者微生物种群失调时引起炎症等疾病,因此可以通过测量由实施例2中制备的枯草芽孢杆菌BS168-AFP1-S对白色念珠菌的抑制效果验证所述融合蛋白的功能,本发明使用的菌株为白色念珠菌标准菌株ATCC90028(购于美国ATCC细胞库),具体操作步骤如下:
(1)将购买的白色念珠菌菌种接种到LB液体培养基中,37℃,200 rpm,培养12 h,使用LB培养基稀释培养得到的白色念珠菌菌液至OD600值为0.8得到白色念珠菌实验菌液;
(2)将白色念珠菌实验菌液接种于96孔板中,每个孔槽接种200 μL;
(3)将枯草芽孢杆菌BS168-AFP1-S接种于孔槽中,每个空槽接种10μL;
(4)37℃静置培养,于18 h测量OD600值;
设置的处理为:AFP1原液+白色念珠菌、AFP1 LB稀释2倍+白色念珠菌、AFP1 LB稀释4倍+白色念珠菌、AFP1 LB稀释8倍+白色念珠菌、AFP1 LB稀释16倍+白色念珠菌,以LB+白色念珠菌为空白对照,单个处理设置7个重复,实验重复3次。
抑菌效果判定标准:以阴性对照孔OD600值为参照,高于此值的孔槽为促进抑菌生长,低于此值的孔槽为抑菌细菌生长,具有抑菌活性。
如图6所示,表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的枯草芽孢杆菌BS168-AFP1-S分泌的融合蛋白溶液可以抑制白色念珠菌,并且稀释4倍后仍然具备较高的抑制效果。
实施例7
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白对铜绿假单胞菌抑菌效果分析
铜绿假单胞菌多生存在动物皮肤、肠道中,在禽畜免疫力下降或者微生物种群失调时引起炎症等疾病,因此可以通过测量由实施例2中制备的枯草芽孢杆菌BS168-AFP1-S对铜绿假单胞菌的抑制效果验证所述融合蛋白的功能,本发明使用的菌株为铜绿假单胞菌标准菌株ATCC15692(购于美国ATCC细胞库),具体操作步骤如下:
(1)将购买的铜绿假单胞菌菌种接种到LB液体培养基中,37℃,200 rpm,培养12h,使用LB培养基稀释培养得到的铜绿假单胞菌菌液至OD600值为0.8得到铜绿假单胞菌实验菌液;
(2)将铜绿假单胞菌实验菌液接种于96孔板中,每个孔槽接种200 μL;
(3)将枯草芽孢杆菌BS168-AFP1-S接种于孔槽中,每个空槽接种10μL;
(4)37℃静置培养,于18 h测量OD600值;
设置的处理为:AFP1原液+铜绿假单胞菌、AFP1 LB稀释2倍+铜绿假单胞菌、AFP1LB稀释4倍+铜绿假单胞菌、AFP1 LB稀释8倍+铜绿假单胞菌、AFP1 LB稀释16倍+铜绿假单胞菌,以LB+铜绿假单胞菌为空白对照,单个处理设置7个重复,实验重复3次。
抑菌效果判定标准:以阴性对照孔OD600值为参照,高于此值的孔槽为促进抑菌生长,低于此值的孔槽为抑菌细菌生长,具有抑菌活性。
如图7所示,表达植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的枯草芽孢杆菌BS168-AFP1-S分泌的融合蛋白溶液可以抑制铜绿假单胞菌,并且稀释4倍后仍然具备较高的抑制效果。
实施例8
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白应用于畜类动物
选取断奶仔猪450头,分为试验组和对照组,试验组150头,对照组150头,阳性对照组150头,试验组将表达抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ的融合蛋白的枯草芽孢杆菌BS168-AFP1-S按照0.5 kg/吨的量加入常规日粮中,其中枯草芽孢杆菌BS168-AFP1-S菌株的含量为1000亿CFU/g,阳性对照组为常规日粮与抗生素阿莫西林100 g/吨混合后进行喂养,空白对照组按常规日粮喂养,在试验期间所有猪按正常的免疫程序进行疫苗接种,并按常规方式进行管理,试验阶段从35日龄至64日龄,共30天,然后测量平均日增重、死亡率、腹泻率,其中死亡率为死亡仔猪数量占测试总仔猪数量的百分数,腹泻率为腹泻仔猪数量占测试总仔猪数量的百分数。
表1 使用植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白防治畜类动物细菌性感染
如表1所示,使用枯草芽孢杆菌BS168-AFP1-S喂养仔猪可以提高平均日增重并且降低死亡率与腹泻率,因此结果表明植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白可以应用于禽类动物预防或者治疗细菌性感染。
实施例9
植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白应用于禽类动物
针对同一鸡舍的350日龄的产蛋鸡进行拌料饲喂,分为试验组和对照组,试验组为3000只,对照组为3000只,试验组添加本发明制备的枯草芽孢杆菌BS168-AFP1-S,添加量为300克/吨,对照组不添加枯草芽孢杆菌BS168-AFP1-S,按同一正常日粮和饲喂程序,每天观察二次,时间为早晚各一次,全程基础日粮和其它饲养管理、饲养环境、饲养员、免疫保健等实验组与对照组保持相同,试验时间为40天。
表2 植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白对产蛋鸡肠道的影响
结合表2和试验过程的观察发现,在试验的开始时试验组和对照组均存在一样的稀便,但是试验组在第六天粪便就开始好转,在试验结束时粪便基本上没有稀便,而对照组在试验结束时反而严重度增加;此外,试验组和对照组在试验前产蛋率都在82-83%,试验结束时试验组的产蛋率明显提高;同时,采食量也存在变化具体表现为:试验组在第10天时采食量下降,由118克/天下降为116克/天,然而对照组不但没有下降,反而有所上升,由116克/天增加为122克/天,因此试验组明显降低了饲料损耗,进而降低生产成本,提高综合效益。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
以上对本发明及其实施方式进行了描述,这种描述没有限制性,附图中所示的也只是本发明的实施方式之一,实际的应用并不局限于此。总而言之如果本领域的普通技术人员受其启示,在不脱离本发明创造宗旨的情况下,不经创造性的设计出与该技术方案相似的方式及实施例,均应属于本发明的保护范围。
序列表
<110> 五康生物科技股份有限公司
<120> 一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白及应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 258
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgattcaaa aacgaaagcg gacggacagt ttcgttcaga cttgtgctta tgtgcacgct 60
gttattgtca gtttgccgat tacaaaaaca tcagccgatg gcgttaaact ttgcgatgtt 120
ccttctggca catggtctgg ccattgcggc tcttcttcta aatgctctca acaatgcaaa 180
gatcgtgaac atttcgctta cggcggcgct tgccattacc aattcccttc tgttaaatgc 240
ttctgcaaac gtcaatgc 258
<210> 2
<211> 86
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ile Gln Lys Arg Lys Arg Thr Asp Ser Phe Val Gln Thr Cys Ala
1 5 10 15
Tyr Val His Ala Val Ile Val Ser Leu Pro Ile Thr Lys Thr Ser Ala
20 25 30
Asp Gly Val Lys Leu Cys Asp Val Pro Ser Gly Thr Trp Ser Gly His
35 40 45
Cys Gly Ser Ser Ser Lys Cys Ser Gln Gln Cys Lys Asp Arg Glu His
50 55 60
Phe Ala Tyr Gly Gly Ala Cys His Tyr Gln Phe Pro Ser Val Lys Cys
65 70 75 80
Phe Cys Lys Arg Gln Cys
85
Claims (10)
1.一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白,其特征在于:所述融合蛋白的核苷酸序列如SEQ ID NO:1所示,所述融合蛋白的氨基酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述的一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白,其特征在于:所述融合蛋白的表达宿主为大肠杆菌、毕赤酵母、枯草芽孢杆菌中的一种。
3.一种根据权利要求1所述的植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体,其特征在于:所述表达载体包含所述融合蛋白的核苷酸。
4.一种根据权利要求3所述的植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体的构建方法,其特征在于:所述构建方法包括如下步骤:
步骤一:以人工合成的基因AFP1-S为模板,使用引物进行PCR扩增得到带有酶切位点的AFP1-S片段;
步骤二:使用限制性内切酶切割载体与AFP1-S片段,采用同源重组酶连接载体与AFP1- S片段得到连接产物;
步骤三:将所述连接产物转化大肠杆菌感受态中获得阳性克隆子,然后提取所述阳性克隆子的质粒,经过DNA测序后,得到所述融合蛋白的表达载体。
5.根据权利要求4所述的一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体的构建方法,其特征在于:所述载体为pWUKANG01,所述融合蛋白的表达载体为pWUKANG01-AFP1-S。
6.根据权利要求5所述的一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体的构建方法,其特征在于:所述限制性内切酶为XholⅠ。
7.根据权利要求6所述的一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的表达载体的构建方法,其特征在于:所述引物为5’-GCGGTACCGAGCTCGCTCGAG-3’
和5’-TGCAGCGGCTAGCCCCTCGAGTCA-3’。
8.一种根据权利要求1所述的植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的应用,其特征在于:所述融合蛋白应用于构建预防和治疗禽畜细菌性腹泻的工程菌株。
9.根据权利要求8所述的一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的应用,其特征在于:所述融合蛋白应用于制备预防和治疗禽畜细菌性腹泻的药物。
10.根据权利要求9所述的一种植物抗菌肽AFP1与芽孢杆菌分泌肽SPamyQ融合蛋白的应用,其特征在于:所述细菌性腹泻包括由沙门氏菌、大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌和白色念珠菌的一种或者多种引起的腹泻。
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