CN116785458A - 半滑舌鳎抗病蛋白shp-1基因的应用 - Google Patents
半滑舌鳎抗病蛋白shp-1基因的应用 Download PDFInfo
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Abstract
本发明属于鱼类基因工程技术领域，具体涉及一种半滑舌鳎抗病蛋白SHP‑1基因的应用。本发明提供了半滑舌鳎抗病蛋白SHP‑1基因在制备用于防治半滑舌鳎细菌病的产品中的应用，所述SHP‑1基因的核苷酸序列如SEQ ID NO：1所示，所述蛋白SHP‑1的氨基酸序列如SEQ IDNO：2所示，可防治半滑舌鳎的细菌性炎症，抑制炎症细胞因子的产生，调控NFκB和IL‑6‑JAK‑STAT3信号通路，阻断细胞因子风暴的产生，能显著降低水产养殖中病原菌感染导致的经济损失，对水产养殖业发展至关重要。
Description
技术领域
本发明属于鱼类基因工程技术领域，具体涉及一种半滑舌鳎抗病蛋白SHP-1基因的应用。
背景技术
半滑舌鳎(Cynoglossus semilaevis)其因肉质鲜美，营养丰富，是我国沿海城市主要养殖的高经济鱼类。鳗弧菌(Vibrio anguillarum)是一种条件致病菌，是导致水产养殖鱼类发生弧菌病的主要病原菌，鳗弧菌的感染途径主要包括皮肤、鳃、肠道等组织器官，能够引起水产动物全身性的组织病变，引起剧烈的炎症反应，从而产生大量的促炎介质并导致细胞因子风暴的发生，使鱼类在急性感染期发生大面积的死亡。因此，在急性感染早期阻断细胞因子风暴的产生对挽救水产养殖业的损失至关重要。
活化B细胞核因子kappa轻链增强子(Nuclear factor kappa light chainenhancer of activated B cells，NFκB)是一种广泛存在的转录因子，在先天免疫应答中发挥重要作用。NFκB是一个二聚体转录因子家族，主要由NF-κB2 p52/p100、NF-κB1 p50/p105、c-Rel、RelA/p65和RelB组成，这些蛋白形成异二聚体或同二聚体复合物，可调控基因表达，并影响先天和适应性免疫、炎症、应激反应、B细胞发育和淋巴器官形成等各种不同的生物学过程。研究发现，病原菌感染会导致NFκB刺激触发炎症介质的表达，包括细胞因子、趋化因子和胞粘附分子，并形成正反馈回路，可以在短时间内产生大量的炎症因子，导致细胞因子风暴的发生，短时间大量的炎症因子的产生会损伤机体的组织与器官，从而导致机体因过度的炎症反应而死亡。
SHP-1(src-homology domain 2-containing protein tyrosine phosphatase-1,SHP-1)，是由PTPN6基因编码的含有SH2结构域的非受体型蛋白酪氨酸磷酸酶。我们通过研究发现，SHP-1作为JAK/STAT3、NFκB以及PI3K/AKT多种信号通路的负调控因子，其去磷酸化作用影响细胞增殖、分化、活性和凋亡，SHP-1可能与硬骨鱼类的炎症反应具有显著的相关性，通过对SHP-1在鱼类炎症方面作用机制的研究将降低水产养殖中病原菌感染导致的经济损失，对水产养殖业发展至关重要。
发明内容
本发明的目的在于提供一种半滑舌鳎抗病蛋白SHP-1基因在提高硬骨鱼类抗病力方面的应用，所述SHP-1基因的表达可防治鱼类的细菌性炎症，抑制炎症细胞因子的产生，阻断细胞因子风暴的产生。
本发明是采用以下技术方案实现的：
本发明提供了半滑舌鳎抗病蛋白SHP-1基因在制备用于防治半滑舌鳎细菌病的产品中的应用。
本发明提供了半滑舌鳎抗病蛋白SHP-1在制备用于防治半滑舌鳎细菌病的产品中的应用。
本发明还提供了半滑舌鳎抗病蛋白SHP-1基因在硬骨鱼类抗病新家系育种中的应用。
所述SHP-1基因的核苷酸序列如SEQ ID NO：1所示，所述蛋白SHP-1的氨基酸序列如SEQ ID NO：2所示。
所述产品具有下述1)-4)种至少一种功能：
1)防治半滑舌鳎的细菌性炎症；
2)抑制炎症细胞因子的产生；
3)调控NFκB和IL-6-JAK-STAT3信号通路；
4)阻断细胞因子风暴的产生。
所述产品包括半滑舌鳎抗病蛋白SHP-1或含SHP-1基因的重组表达质粒。
所述产品包括饲料、饲料添加剂、药物或药物组合物。
所述含SHP-1基因的重组表达质粒的制备方法，包括以下步骤：
(1)提取半滑舌鳎总RNA，逆转录得到总cDNA；
(2)设计SHP-1基因的正向引物和反向引物，以半滑舌鳎总cDNA为模板扩增得到扩增产物；
(3)将扩增产物进行分子克隆，得到重组表达质粒。
优选的，分子克隆的酶切位点为HindIII和BamHI；
正向引物序列为CCAAGCTTATGGTTCGGTGGTTCCAC；
反向引物序列为CGGGATCCTTTTTTTCGCAACGAGCCGC。
与现有技术相比，本发明具有以下有益效果：
1、本发明所述SHP-1基因的表达可防治鱼类的细菌性炎症，抑制炎症细胞因子的产生，调控NFκB和IL-6-JAK-STAT3信号通路，阻断细胞因子风暴的产生，能显著降低水产养殖中病原菌感染导致的经济损失，对水产养殖业发展至关重要。
2、本发明所述SHP-1基因可通过基因编辑技术建立新的鱼类抗病家系。
附图说明
图1、pcDNA3.1-SHP-1-EGFP重组表达质粒结构示意图；
图2、SHP-1扩增、双酶切及菌落PCR鉴定；
图3、高浓度组半滑舌鳎死亡率统计和生存分析曲线；
图4、低浓度组半滑舌鳎死亡率统计和生存分析曲线；
图5、高浓度组半滑舌鳎SHP-1、NFκB以及JAK-STAT3信号通路关键因子的表达情况；
图6、高浓度组半滑舌鳎HE染色分析半滑舌鳎肝组织病理损伤变化；
图7、高浓度组半滑舌鳎HE染色分析半滑舌鳎肠组织病理损伤变化。
具体实施方式
下面结合实施例对本发明作进一步描述。
需要说明的是，实施例中用到的所有原料除特殊说明外，均为市购，其中，引物由北京擎科生物科技有限公司合成；SYBR Green Mix混合物购自南京诺唯赞生物公司；苏木素、伊红染料、中性树胶购自武汉塞维尔生物科技有限公司。
实施例1
制备SHP-1重组表达质粒pcDNA3.1-SHP-1-EGFP，包括以下步骤：
(1)提取半滑舌鳎肝组织总RNA，逆转录得到总cDNA；
(2)设计SHP-1基因的正向引物和反向引物，以半滑舌鳎总cDNA为模板PCR扩增得到扩增产物；
所述正向引物序列为CCAAGCTTATGGTTCGGTGGTTCCAC；
所述反向引物序列为CGGGATCCTTTTTTTCGCAACGAGCCGC；
PCR反应体系：2×Taq酶预混液10μL，上游引物1μL，下游引物1μL，cDNA 1μL，ddH2O7μL；
PCR扩增程序：95℃预变性5min，95℃变性30s，55℃退火30s，72℃延伸1min，35个循环。
(4)将扩增产物进行分子克隆，得到重组表达质粒；
以HindIII和BamHI为酶切位点将扩增产物SHP-1插入到pcDNA3.1EGFP载体中构建pcDNA3.1-SHP-1-EGFP重组表达质粒，并通过菌落PCR及双酶切进行鉴定。构建质粒模式图如图1；成功扩增SHP-1基因、双酶切及菌落PCR鉴定如图2。
实施例2
选取重量10±5g、体长10±5cm的半滑舌鳎作为实验对象，分为3组，对照组、SHP-1高表达组和抑制剂组，对照组静脉注射PBS，SHP-1高表达组静脉注射2μg/g的SHP-1重组表达质粒，提高SHP-1的表达水平，抑制剂组静脉注射3μg/g的TPI-1，TPI-1为SHP-1小分子抑制剂，抑制SHP-1的表达水平。
饲养48h后进行攻毒实验，腹腔注射鳗弧菌，模拟水产养殖的细菌感染，每组半滑舌鳎分别随机平均分为高浓度组和低浓度组，高浓度组半滑舌鳎腹腔注射3.67×106cfu/g鳗弧菌，低浓度组腹腔注射1.3×106cfu/g鳗弧菌。
统计高浓度组和低浓度组在注射前0h以及注射后6h、12h、24h、48h、72h的半滑舌鳎死亡率和生存曲线分析，结果如图3，高浓度组的对照组(Control)死亡率为85％，SHP-1高表达组(SHP-1)死亡率为61.67％，抑制剂组(Inhibition)死亡率为86.67％；如图4，低浓度组的对照组组死亡率为43.33％，SHP-1高表达组死亡率为6.67％，抑制剂组死亡率为40％。由此可以看出，无论高浓度还是低浓度鳗弧菌感染，SHP-1高表达组死亡率均为最低，且通过使半滑舌鳎SHP-1高表达而降低其感染时死亡率的效果显著。
实施例3
对实施例2中高浓度组进行下一步研究，分别采集三组注射鳗弧菌前0h以及注射后12h、24h、48h、72h的肝组织和肠组织样品。
1、对肝组织样品进行qPCR验证SHP-1及IL-1b、MYD88、NFκB1、IL-6、JAK2、STAT3、TLR3、TLR5的转录表达；
qPCR反应体系：2×SYBR酶预混液10μL，上游引物0.4μL，下游引物0.4μL，cDNA1μL，ddH2O 8.2μL，qPCR特异性引物如表1；
qPCR反应程序：95℃预变性30s，95℃变性10s，60℃退火30s，40个循环。
qPCR结果如图5，NFκB以及IL-6-JAK-STAT3是重要的炎症信号通路，通过调控这两种信号通路可以有效控制机体的炎症，从而避免细胞因子风暴的产生，使机体因过度的免疫反应而迅速死亡。IL-1b和IL-6作为重要的促炎细胞因子，分别是NFκB以及JAK-STAT3信号通路的上游结合因子，二者之间可以互相调控，从而影响机体的炎症进程。通过高表达SHP-1后可以明显抑制促炎因子IL-1b和IL-6的表达，并可以调控NFκB以及JAK-STAT3信号通路。qPCR结果证明，鳗弧菌感染后12h与0h相比，对照组和抑制剂组促炎细胞因子IL-1b、IL-6、NFκB1表达量显著上升，SHP-1高表达组的促炎细胞因子显著下降。说明SHP-1可以明显抑制促炎细胞因子的表达，并可抑制炎症信号通路的激活与转导。
表1 qPCR特异性引物
2、对肝组织样品和肠组织样品进行HE染色，检测半滑舌鳎肝和肠的组织病理变化。将采集的组织样品切成0.5×0.5cm的组织块，并加入4％多聚甲醛固定，将固定好的组织样品进行石蜡包埋，并使用石蜡切片机将石蜡块切成厚度为4-8μm的切片，将切片进行脱蜡，分别进行苏木素和伊红染色，染色后使用中性树脂进行封片，通过显微镜成像得到组织病理光镜图，肝组织光镜图结果如图6，肠组织光镜图结果如图7，图中，代表炎细胞浸润，代表肠粘膜层损伤断裂。
从图中可以看出，抑制剂组的肝和肠组织损伤程度明显高于对照组，SHP-1高表达组肝和肠的组织损伤程度显著低于对照组，高表达SHP-1在鳗弧菌感染后表现出对半滑舌鳎肝、肠组织良好的保护作用。
Claims (10)
1.半滑舌鳎抗病蛋白SHP-1基因在制备用于防治半滑舌鳎细菌病的产品中的应用。
2.半滑舌鳎抗病蛋白SHP-1在制备用于防治半滑舌鳎细菌病的产品中的应用。
3.半滑舌鳎抗病蛋白SHP-1基因在硬骨鱼类抗病新家系育种中的应用。
4.根据权利要求1-权利要求3所述的应用，其特征在于：所述SHP-1基因的核苷酸序列如SEQ ID NO：1所示，所述蛋白SHP-1的氨基酸序列如SEQ ID NO：2所示。
5.根据权利要求4所述的应用，其特征在于：所述产品具有下述1)-4)种至少一种功能：
1)防治半滑舌鳎的细菌性炎症；
2)抑制炎症细胞因子的产生；
3)调控NFκB和IL-6-JAK-STAT3信号通路；
4)阻断细胞因子风暴的产生。
6.根据权利要求5所述的应用，其特征在于：所述产品包括半滑舌鳎抗病蛋白SHP-1或含SHP-1基因的重组表达质粒。
7.根据权利要求6所述的应用，其特征在于：所述产品包括饲料、饲料添加剂、药物或药物组合物。
8.根据权利要求6所述的应用，其特征在于：所述含SHP-1基因的重组表达质粒的制备方法，包括以下步骤：
(1)提取半滑舌鳎总RNA，逆转录得到总cDNA；
(2)设计SHP-1基因的正向引物和反向引物，以半滑舌鳎总cDNA为模板扩增得到扩增产物；
(3)将扩增产物进行分子克隆，得到重组表达质粒。
9.根据权利要求8所述的应用，其特征在于：分子克隆的酶切位点为HindIII和BamHI。
10.根据权利要求8所述的应用，其特征在于：
正向引物序列为CCAAGCTTATGGTTCGGTGGTTCCAC；
反向引物序列为CGGGATCCTTTTTTTCGCAACGAGCCGC。
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