CN117587024A - 一种绵羊抗传染性胸膜肺炎的s100a9基因及其应用 - Google Patents
一种绵羊抗传染性胸膜肺炎的s100a9基因及其应用 Download PDFInfo
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Abstract
本发明属于生物领域，本发明提供了一种绵羊抗传染性胸膜肺炎的相关基因S100A9及其应用。本发明发现，在侵染有绵羊肺炎支原体的中性粒细胞中过表达S100A9基因，能够增强中性粒细胞对支原体的清除，使绵羊肺炎支原体的增殖水平降低，表明S100A9基因可提高绵羊中性粒细胞对支原体的清除能力，提高绵羊对肺炎支原体的抵抗能力，用于后期抗病品种选育,可显著缩短改良品种所需的时间。
Description
技术领域
本发明属于生物领域，本发明提供了一种绵羊抗传染性胸膜肺炎的相关基因S100A9及其应用。
背景技术
绵羊肺炎支原体(Mycoplasma ovipneumoniae，MO)是绵羊和山羊呼吸道中最常见的支原体，能引起感染羊只尤其是羔羊增生性间质性肺炎的病原体。患病羊临床上主要表现为发热、咳嗽、消瘦。病程可达数月以上，导致出栏率下降造成大量的经济损失。本病的传播方式主要通过飞沫传播，患病羊咳嗽后会将病原体散播在空气当中引发羊群大规模感染。近年来，随着国内集约化养殖业的发展，绵羊肺炎支原体在全国各省市大型养殖场不断流行和蔓延，致病性增强，危害加重。但目前其致病机制和免疫机制尚不完全清楚。
中性粒细胞是血液中最丰富的免疫细胞，作为宿主抵抗入侵病原体的第一道防线，中性粒细胞具备固有的吞噬能力，可通过释放黏附抗菌蛋白，蛋白水解酶以及活性氧杀灭病原体，其表面表达多种趋化因子受体和补体受体，具有调节炎性反应的功能。在我们前期试验中发现，动物感染绵羊肺炎支原体后肺部中性粒细胞数量明显改变，说明其参与到机体抵抗外来支原体反应当中。
S100A9(也称为MRP14)是属于S100家族的Ca2+结合蛋白，参与细胞骨架重排和花生四烯酸代谢，于1965年首次作为神经蛋白从牛大脑中提取。S100A9具有带电荷氨基酸残基的螺旋-环-螺旋基序，这是S100家族成员的共同特征，导致它对二价离子(如Ca2+和Zn2+)具有高亲和力。与二价离子的结合后改变其构象，这是发挥其相应功能的基础。在炎症过程中S100A9被释放，并通过刺激白细胞募集和诱导细胞因子分泌在调节炎症反应中发挥关键作用。S100A9能通过Ca2+和p38 MAPK信号的磷酸化逆转微管的形成，并导致细胞骨架重排，从而导致白细胞的有效迁移。且在胞外，纯化的S100A9能够抑制多种微生物的生长，包括大肠杆菌、白色念珠菌、金黄色葡萄球菌、肺炎克雷伯菌、鼠伤寒沙门氏菌等，但对绵羊肺炎支原体的具体作用未见报道。
本发明发现，在侵染有绵羊肺炎支原体的中性粒细胞中过表达S100A9基因，能够增强中性粒细胞对支原体的清除，使绵羊肺炎支原体的增殖水平降低，表明S100A9基因可提高绵羊中性粒细胞对支原体的清除能力，提高绵羊对肺炎支原体的抵抗能力，用于后期抗病品种选育,可显著缩短改良品种所需的时间。
发明内容
针对背景技术所提出的技术问题，本发明的目的在于提供S100A9基因、含有S100A9基因的生物材料，或S100A9蛋白的应用。
本发明的目的可以通过以下技术方案实现：
第一方面，本发明请求保护具有如SEQ ID NO.1所示核苷酸序列的S100A9基因在(1)或(2)或(3)或(4)中的应用：
(1)提高绵羊中性粒细胞对肺炎支原体的清除能力；
(2)提高绵羊对肺炎支原体的抵抗能力；
(3)提高绵羊对传染性胸膜肺炎的抗性；
(4)培育对传染性胸膜肺炎的抗性提高的绵羊抗病新品种。
第二方面，本发明请求保护含有上述S100A9基因的生物材料在(1)或(2)或(3)或(4)中的应用：
(1)提高绵羊中性粒细胞对肺炎支原体的清除能力；
(2)提高绵羊对肺炎支原体的抵抗能力；
(3)提高绵羊对传染性胸膜肺炎的抗性；
(4)培育对传染性胸膜肺炎的抗性提高的绵羊抗病新品种。
进一步，所述的生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
进一步，上述的应用为，在绵羊中性粒细胞中过表达所述的S100A9基因，能够增强中性粒细胞对肺炎支原体的清除能力，提高绵羊对肺炎支原体的抵抗能力。
更进一步，在绵羊中性粒细胞中过表达所述的S100A9基因的过程为：将所述的S100A9基因插入到表达载体pCMV-HA-C中构建S100A9基因的过表达载体pCMV-S100A9-HA；将构建的过表达载体pCMV-S100A9-HA转染绵羊中性粒细胞得到过表达S100A9基因的绵羊中性粒细胞。
本发明的具体实施方式中，使用5`EcoRI-3`KpnI双酶切体系，将S100A9基因插入到表达载体pCMV-HA-C中。
第三方面，本发明请求保护由上述S100A9基因编码的S100A9蛋白在制备用于预防或治疗绵羊肺炎支原体感染的药物或佐剂中的应用。
进一步，所述S100A9蛋白的氨基酸序列如SEQ ID NO.2所示。
本发明的有益效果是：
本发明发现，在侵染有绵羊肺炎支原体的中性粒细胞中过表达S100A9基因，提高S100A9蛋白的含量，能够增强中性粒细胞对支原体的清除能力，使绵羊肺炎支原体的增殖水平降低，表明S100A9蛋白可用于制备预防或治疗绵羊肺炎支原体感染的药物或佐剂，用于抑制绵羊肺炎支原体的增殖。
附图说明
图1是本发明实施例提供的S100A9蛋白同源性对比分析示意图；
图2在感染支原体的中性粒细胞中，随着S100A9质粒转染量的增加，S100A9基因含量逐渐增加结果图。
图3在感染支原体的中性粒细胞中，随着S100A9质粒转染量的增加，S100A9蛋白含量逐渐增加结果图。
图4在感染支原体的中性粒细胞中，随着S100A9质粒转染量的增加，支原体拷贝数逐渐降低的结果图。
具体实施方式
下面结合实施例和相关附图对本发明的技术方案进行进一步详细说明。但值得注意的是，本发明的实施方式不仅限于此，相关人员可以在不违背本发明的宗旨和精神的情况下通过其他不同的形式来实现。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，均自常规生化试剂公司购买获得或为本领域技术人员公知的常规生物材料。
实验材料：
pCMV-HA-C载体和pMD18T载体购自南京擎科生物科技有限公司。
绵羊肺炎支原体临床分离株NJ01株由江苏省农业科学院兽医研究所刘茂军研究员提供。绵羊肺炎支原体(Mycoplasma ovipneumoniae)NJ01株，已保藏在中国典型培养物保藏中心，保藏号为CCTCC NO：M 2020907，保藏日期为2020年12月14日。
绵羊中性粒细胞是使用北京索莱宝科技有限公司人外周血中性粒细胞分离液试剂盒从绵羊外周血中获得。
实施例1S100A9基因的过表达载体构建
发明人前期从感染绵羊肺炎支原体的单细胞测序结果发现，S100A9基因在感染羊只的中性粒细胞中差异性表达，因此推测S100A9基因可能参与中性粒细胞清除支原体的过程。S100A9蛋白同源性对比分析示意图如图1所示。
将绵羊S100A9基因两端加上EcoRI和KpnI酶切位点，S100A9基因核苷酸序列如SEQID NO.1,所示，由南京擎科生物科技有限公司进行全基因合成，并且连入载体质粒pCMV-HA-C中，得到重组质粒pCMV-S100A9-HA，后将重组质粒转化至大肠杆菌DH5α感受态细胞，挑取单个菌落接种于含有氨苄青霉素的LB液体培养基中，37℃振荡培养后使用去内毒素质粒提取试剂盒提取质粒。
构建含有支原体Tuf基因片段的载体，以支原体NJ01株基因组为模板，使用引物(F：ATGTTGACCACGGAAAAACC；R：GGCATAGTGACGCTTGTCTG)扩增出目的基因片段，并将其连接到pMD18T载体的EcoRI和KpnI酶切位点间，得到重组质粒pMD18T-Tuf，然后将重组质粒转化至大肠杆菌DH5α感受态细胞，挑取单个菌落接种于含有氨苄青霉素的LB液体培养基中，37℃振荡培养后使用质粒提取试剂盒提取质粒。
实施例2不同浓度的S100A9蛋白促进中性粒细胞对支原体的清除
1.过表达S100A9基因的中性粒细胞的制备
使用浓度为100μg/ml的多聚赖氨酸溶液提前包被24孔板放置温箱中过夜，第二天取出培养板在每孔中加入5×105个中性粒细胞，放置在含有5％ CO2的37℃温箱中平衡3小时后，使用Lip3000转染试剂分别转染pCMV-HA-C空白质粒，以及不同剂量的pCMV-S100A9-HA质粒，分别为0.5μg、1μg、2μg。
2.中性粒细胞感染MO样品的制备
转染6小时以后，接种支原体(MOI＝0.1)，同时以空白培养基作为对照，接种后12小时提取上清液中DNA检测支原体拷贝数，用RT-qPCR检测细胞中S100A9基因表达量，用Western Blot测定细胞中S100A9蛋白含量。
其中，S100A9基因的上下游引物分别为：
F:CTGACGGTAGCCTCCCACGA，R:CGCCACCCTTGTCGAAGCC。
内参基因ACTB的上下游引物分别为：
F:ACCATGTACCCTGGCATCGCAGA，R：CCACACGGAGTACTTGCGCTCA。
3.细胞培养液上清中支原体拷贝数的测定
测定上述步骤2中获得的上清中支原体的DNA拷贝数，进行支原体含量分析。
DNA拷贝数测定步骤如下：使用磁珠法DNA提取试剂盒提取上清液中支原体的总DNA留存备用。用核酸蛋白检测仪测定pMD18T-Tuf质粒浓度，单位为ng/μL，按照公式copies/μL＝(6.02×1023)×(浓度ng/μL×10-9)/(DNA length×660)将重组质粒的浓度单位换算成copies/μL。调整质粒母液为1010copies/μL后进行10倍倍比稀释，制备出浓度为102、103、104、105、106、107、108、109、1010copies/μL的重组质粒标准品。使用荧光定量PCR检测标准品及样品的CT值，计算出上清中支原体的总含量。
实验结果如图2-4所示，随着转染质粒总量的增加，S100A9基因含量逐渐增加(图2)，S100A9蛋白含量逐渐增加(图3)，且随着S100A9蛋白含量的增加，支原体的拷贝数降低，呈剂量依赖关系(图4)。
综上，本发明实施例以中性粒细胞为例，在绵羊中性粒细胞中过表达所述的S100A9基因，提高S100A9蛋白的含量，显著提升了中性粒细胞清除绵羊肺炎支原体的效率，提高绵羊对肺炎支原体的抵抗能力，抑制了支原体的增殖，表明本发明所述的S100A9蛋白可用于制备抗绵羊肺炎支原体感染的药物或佐剂。
SEQ ID NO.1：
S100A9基因核苷酸序列：
ATGGCGGACCAATTGTCACAAATGGAATCCAGCATAGAAACCATCATCAACATCTTCCAC
CAGTACTCTATACGGCTAAGGGACCCGGAAACCCTGATCCGGAAAGAACTCAAACAGCT
GGTGCAAAAAGAGCTGCCAAACTTTCTCAAGAAGCAGCATAAGGATGAAGAGGCCATC
AACGAGATCATGGAGGACCTGGACACAAACCAAGACAAGCAGCTGAGCTTCGAGGAG
TTCATTATGCTGGTGGCCAGGCTGACGGTAGCCTCCCACGAGGAGATGCACAAGACCGC
ACCCCCGGGAAGAGGGCACCGGCACGGGCCAGGCTTCGACAAGGGTGGCGCAGGCCC
GTGCCCTGGCCAGGGCGGCCCCGACCACAGCCACGGTGGTCACGGCCACAGCCACGGC
GGTCATGGCCACAGCCACGGCGGTCACGGCCACAGCCAC
SEQ ID NO.2：
S100A9蛋白的氨基酸序列包含有149个残基：
MADQLSQMESSIETIINIFHQYSIRLRDPETLIRKELKQLVQKELPNFLKKQHKDEEAIN
EIMEDLDTNQDKQLSFEEFIMLVARLTVASHEEMHKTAPPGRGHRHGPGFDKGGAGPCPG
QGGPDHSHGGHGHSHGGHGHSHGGHGHSH。
Claims (7)
1.具有如SEQ ID NO.1所示核苷酸序列的S100A9基因在(1)或(2)或(3)或(4)中的应用：
(1)提高绵羊中性粒细胞对肺炎支原体的清除能力；
(2)提高绵羊对肺炎支原体的抵抗能力；
(3)提高绵羊对传染性胸膜肺炎的抗性；
(4)培育对传染性胸膜肺炎的抗性提高的绵羊抗病新品种。
2.含有权利要求1中所述S100A9基因的生物材料在(1)或(2)或(3)或(4)中的应用：
(1)提高绵羊中性粒细胞对肺炎支原体的清除能力；
(2)提高绵羊对肺炎支原体的抵抗能力；
(3)提高绵羊对传染性胸膜肺炎的抗性；
(4)培育对传染性胸膜肺炎的抗性提高的绵羊抗病新品种。
3.根据权利要求2所述的应用，其特征在于，所述的生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
4.根据权利要求1或2所述的应用，其特征在于，在绵羊中性粒细胞中过表达所述的S100A9基因，能够增强中性粒细胞对肺炎支原体的清除能力，提高绵羊对肺炎支原体的抵抗能力。
5.根据权利要求4所述的应用，其特征在于，在绵羊中性粒细胞中过表达所述的S100A9基因的过程为：将所述的S100A9基因插入到表达载体pCMV-HA-C中构建S100A9基因的过表达载体pCMV-S100A9-HA；将构建的过表达载体pCMV-S100A9-HA转染绵羊中性粒细胞得到过表达S100A9基因的绵羊中性粒细胞。
6.由权利要求1中所述S100A9基因编码的S100A9蛋白在制备用于预防或治疗绵羊肺炎支原体感染的药物或佐剂中的应用。
7.根据权利要求6所述的应用，其特征在于，所述S100A9蛋白的氨基酸序列如SEQ IDNO.2所示。
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