CN101224300A - 大肠杆菌OmpA作为动物免疫调节剂的应用 - Google Patents
大肠杆菌OmpA作为动物免疫调节剂的应用 Download PDFInfo
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Abstract
本发明涉及大肠杆菌(E.coli)表达的一种重组外膜蛋白A(outer membrane protein A，OmpA)的免疫调节功能。本发明通过大肠杆菌OmpA基因缺失株的免疫调节作用，纯化大肠杆菌OmpA的免疫增强作用，大肠杆菌OmpA抗血清被动免疫增强作用等试验，提供了大肠杆菌OmpA具有调节动物免疫功能，提高动物抗病能力的功效。
Description
技术领域
本发明涉及大肠杆菌(E.coli)表达的一种外膜蛋白A(outer membraneproteinA，OmpA)的免疫调节功能。
背景技术
免疫调节剂是一种具有功能(化合物、蛋白质、多糖、药物)可专致的或非专致的增加或减少免疫反应，也是说可以是增效剂(佐剂)，或免疫增进剂或免疫抑制剂。
在水产动物养殖中，往往使用各种化学药物和抗生素来控制有关疾病，但长期用药，不仅导致病原菌的抗药性越来越明显，而且使药物残留的危害也日益显现，严重地影响水产品安全。在这些情况下，水产养殖动物免疫调节剂应运而生。免疫调节剂也叫免疫增强剂或免疫刺激剂，当它和抗原一起使用时能刺激动物特异性免疫机制，单独使用时能刺激非特异性免疫机制，提高动物对各种抗原的应答能力，增强机体的防病能力。常用的免疫刺激剂有弗氏完全佐剂、葡聚糖、左旋咪唑、中草药和激素、维生素C和E等。因此，研制新型免疫调节剂对增强动物免疫能力，防制动物疾病具有重要意义。在本发明被公布之前，尚未有任何公开或报道过本专利申请中提及的大肠杆菌OmpA的免疫调节功能。
发明内容
本发明的目的在于提供大肠杆菌OmpA基因编码蛋白(OmpA)作为一种新的免疫调节剂在调节动物免疫，增强动物抗病能力中的应用。
本发明通过采用大肠杆菌OmpA基因缺失株免疫小鼠，Dot-ELISA(斑点酶联试验)检测发现该缺失株仅能使小鼠产生1∶400效价的抗体，而没有缺失该基因的正常对照菌株可以产生大于1∶6400效价的抗体，说明OmpA的缺失使该菌对小鼠的免疫原性明显下降，提示OmpA具有免疫增加功能。同时发现该缺失株刺激产生的抗血清可以识别正常对照菌株的10个蛋白点，而没有缺失该基因的对照组刺激产生的抗血清仅能识别正常对照菌株的4个蛋白点，说明OmpA的缺失使该菌刺激小鼠的显性免疫原发生了改变，提示OmpA具有免疫调节作用。
本发明为进一步证明上述结论，克隆大肠杆菌OmpA基因，进行原核表达，发现OmpA免疫后的小鼠可以显著提高对副溶血弧菌、铜绿假单胞菌或美人鱼发光杆菌等病原菌的抵抗力。
本发明采用被动免疫转移试验，即以OmpA免疫血清注射鲫鱼后，对这些鲫鱼进行副溶血弧菌、铜绿假单胞菌或美人鱼发光杆菌的免疫攻毒试验，再一次证明OmpA免疫血清确能明显提高鲫鱼对这些病原菌的抵抗力。这些系列试验说明大肠杆菌OmpA具有明显的免疫调节功能。
附图说明
图1为Dot-ELISA(斑点酶联试验)检测免疫血清效价
图2为双向免疫印迹法检测免疫血清效价和反应条带
图3为SDS-PAGE(聚丙烯酰胺凝胶电泳)检测表达产物1，试验组2，空载体对照
图4为SDS-PAGE检测表达产物的镍柱尿素纯化
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆实验室手册(2001 by ColdSpring Harbor Laboratory Press)中所述的条件，或按照制造厂商所建议的条件。
实施例1
大肠杆菌OmpA基因缺失株的免疫调节作用
1.细菌培养：大肠杆菌OmpA基因缺失株来源于Nara Institute of Scienceand Technology，Japan。挑取单菌落于LB培养基培养过夜后，再按1∶100接种于LB培养基，培养至OD1.0；离心获得细菌。同时以大肠杆菌OmpA基因缺失株的来源株为对照菌株。
2.免疫小鼠：将买回的小鼠驯养1周，每天早晚两次投喂人工配合饲料。驯养1周后无异常的小鼠分组。取纯化上述制备的细菌为免疫原，腹腔注射免疫小鼠，109/只。注射3次，第1次注射后14天进行第2次注射，7天后进行第3次注射。第3次免疫结束后的第5天取血，Dot-ELISA和免疫蛋白质组学技术检测结果。Dot-ELISA发现，所制备的抗血清效价在OmpA基因缺失株为1∶400，在对照组为1∶6400(图1)。免疫蛋白质组学技术发现，所制备的抗血清效价在OmpA基因缺失株为1∶200，对照组为1∶4000；所检测得到的免疫反应点在OmpA基因缺失株为10个，在对照组为4个(图2)。这些结果说明，OmpA基因缺失对抗体效价和免疫反应点具有明显的调节作用。
实施例2
大肠杆菌OmpA的制备和纯化
在该实施例中，将全长的大肠杆菌OmpA编码序列或片段构建入大肠杆菌蛋白质原核表达载体之中，以达到表达和提纯目的蛋白。
OmpA基因编码序列如下：
1 ttaagcctgc ggctgagtta caacgtcttt gataccttta acttcgatct ctacgcgacg
61 atccggagcc aggcagtcga tcagtgcagc acgctgtttc acgttgtcac aggtgttgcc
121 agtaaccggg ttggattcgc ccataccacg tgcggagatc ttgtctgccg ggataccttt
181 ggagatcagg taatcaacaa cagactgagc acggcgctcg gacagaccct ggttgtaagc
241 gtcagaaccg atgcggtcgg tgtaacccag aacaactacg gaaccgtctt tcggatccag
301 gttgctcagc tggctgtaca gctgatccag agcagcctga ccttccggtt tcagggttgc
361 tttgttgaag ttgaacagaa cgtcagactt cagagtgaag tgcttggtct gtacttccgg
421 tgccggagct ggagccggag caactactgg agctgcttcg ccctgaccga aacggtagga
481 aacacccagg ctcagcatgc cgttgtccgg acgagtgccg atggtgtgtg cgtcaccgat
541 gttgttggtc cactggtatt ccagacgggt agcgatttca ggagtgatcg cgtactcaac
601 accgccagcg aagaccggag aaacgccggt gtcgtggttt ttaccataaa cgttggattt
661 agtgtctgca cgccatacca tgccacccag acgagtgtag atgtccaggt cgtcagtgat
721 tgggtaaccc agtttagcgg tcagttgaac gccctgagct ttgtatgcac cgttttcaac
781 gctgcctttg tacggcatac gacctaacca gtcgtaaccc atttcaaagc caacatacgg
841 gttaacctgg taaccaccaa aagcaccagc gcccagttgg ttttcatggg tcgggccatt
901 gttgttgatg aaaccagtgt catggtactg ggaccagccc agtttagcac cagtgtacca
961 ggtgttatct ttcggagcgg cctgcgctac ggtagcgaaa ccagccagtg ccactgcaat
1021 cgcgatagct gtctttttca t
OmpA氨基酸序列如下：
1 mkktaiaiav alagfatvaq aapkdntwyt gaklgwsqyh dtgfinnngp thenqlgaga
61 fggyqvnpyv gfemgydwlg rmpykgsven gaykaqgvql taklgypitd dldiytrlgg
121 mvwradtksn vygknhdtgv spvfaggvey aitpeiatrl eyqwtnnigd ahtigtrpdn
181 gmlslgvsyr fgqgeaapvv apapapapev qtkhftlksd vlfnfnkatl kpegqaaldq
241 lysqlsnldp kdgsvvvlgy tdrigsdayn qglserraqs vvdyliskgi padkisargm
301 gesnpvtgnt cdnvkqraal idclapdrrv eievkgikdv vtqpqa
1、大肠杆菌OmpA基因的扩增：
根据NCBI公布的大肠杆菌K12的基因序列，设计一对引物。引物序列如下：Sense Primer：5’-ccc gaa ttc atg aaa aag aca gct atc-3’；Anti-sense Primer：5’-gggctc gag tta agc ctg cgg ctg a-3’。为便于克隆和表达载体的构建，在引物上分别引入限制性内酶位点EcoR I和Xho I。引物由大连宝生物工程公司合成。PCR反应条件的确立：以热变性法获得的大肠杆菌K12全基因组为模板，在25
l反应体系中加PCR反应缓冲液2.5
l，10mmol/L dNTP1l，5′和3′引物各10umol/L，模板DNA 1-2
l，补水至25
l。PCR循环参数为：94℃，2min，然后进行30个PCR循环(94℃变性30s，60℃退火30s，72℃延伸30s)后继续在72℃延伸10min，4℃保存。用琼脂糖胶电泳来鉴定DNA片断的长度并回收DNA片断。
2、大肠杆菌OmpA基因的克隆、筛选和鉴定：
用限制性内酶EcoR I和Xho I分别酶切DNA片断和PET-HIS(Novagen)表达载体，参照Takara公司的使用说明书，在10
l反应体系中加入solution I 5
l，胶回收的-PCR产物1
l，载体载体0.5
l，补水至10l，16℃左右保温14-16h。连接产物转化E.coli DH5α感受态，涂于含Amp(50ug/ml)的LB固体平板上，37℃培养12～16小时。随机挑取含Amp的LB平板上的重组子，接种于含Amp(50ug/ml)的LB液体培养基上，同时接种含有空PET-HIS质粒的DH5α，37℃培养12～16小时后，用碱裂解法分别提取质粒进行双酶切验证。将有插入的重组子送至上海生工进行序列测定，结果证明与数据库公布序列完全一致。
3、大肠杆菌OmpA基因的表达：将正确的重组质粒以热激法转化大肠杆菌BL21表达菌，37℃培养12～16小时。接种单菌落于含Amp的LB液体培养基中，同时接种含有PET-HIS质粒的BL21作为对照，于30℃培养至OD值0.6，加入IPTG 100
g/ml 35℃诱导两个小时，离心收集菌体，用SDS-PAGE检测插入片段是否表达蛋白，以及表达蛋白的分子量大小，结果如图4。接着，鉴定蛋白质表达产物可溶不可溶鉴定。具体过程如下：将离心后沉淀重悬于1/10体积的PBS或50mmol/L Tris缓冲液(pH8.0)中；加入溶菌酶至100ug/ml，保温30min；反复冻融数次以破坏细胞壁，用超声波处理剪切DNA(超声波的强度以不能产生气泡为适宜；裂解的时间根据实际情况掌握，一般将云雾状的细菌悬浮物裂解至比较清朗即可)；离心(12,000rpm)15min后将培养物分为可溶部分和不溶部分；将15ul上清与4ul 4×SDS样品缓冲液混合，将沉淀物重悬于1×SDS样品缓冲液；将表达样品(上清及沉淀)和非表达对照物进行SDS-PAGE电泳并以考马斯亮蓝染色以便在预计分子量范围内观察表达蛋白带。这一步骤将确定蛋白究竟表达在可溶组分抑或是不溶组分(包涵体)中。经鉴定表达产物为可溶蛋白。
4、大肠杆菌OmpA的纯化：
大量培育菌体，离心后超声处理，离心收集上清用Ni-NTA His Bind亲和柱进行纯化。采用pH5.9(buffer F)和pH4.5(buffer G)两个不同pH值的缓冲液纯化蛋白。用冲洗缓冲液buffer E(8M尿素；0.1M NaH2PO4；10mmTris-Hcl，pH 6.3)洗涤5次，每次1ml；用洗脱缓冲液buffer F(8M尿素；0.1M NaH2PO4；10mmTris-Hcl，pH 5.9)洗脱目的蛋白4次，每次1ml，收集洗脱液；用洗脱缓冲液buffer G(8M尿素；0.1M NaH2PO4；10mmTris-Hcl，pH 4.5)洗脱蛋白4次，每次1ml，收集洗脱液。取收集液进行SDS-PAGE分析。500ml培养液获得洗脱液5管，每管1ml，取15ul上样量，其纯化结果如图3。纯化蛋白进行紫外检测，回收率约在50-70％之间。
实施例3
大肠杆菌OmpA的免疫增强作用：
1.小鼠免疫：将买回的小鼠驯养1周，每天早晚两次投喂人工配合饲料。驯养1周后无异常的小鼠分组。取纯化OmpA与等体积完全佐剂(羊毛脂∶石蜡油＝1∶4v/v，卡介苗为30mg/ml)混合，用注射器反复抽吸后，超声处理2min，即为制备好的抗原，腹腔注射免疫小鼠，80-100μg/只，对照组不加免疫处理。注射3次，每次间隔10天，实验组第2次和第3次使用不完全佐剂与目的纯化蛋白混合后免疫注射。第3次免疫结束后的第5天取血，Dot-ELISA法检测血清免疫效价。
2.血清效价测定：采用Dot-ELISA技术，测定攻毒前小鼠血清免疫效价，结果为1∶800-1000。
3.攻毒保护实验：于免疫结束后的第5天分别腹腔注射50倍半数致死量的副溶血弧菌、铜绿假单胞菌或美人鱼发光杆菌活菌液进行攻毒保护试验。
4.免疫保护作用评估：分别用50倍半数致死量的相应细菌进行攻毒保护性实验，观察其死亡率，根据相对免疫保护率来比较分析这些免疫原性蛋白质的保护效果，结果发现对副溶血弧菌、铜绿假单胞菌或美人鱼发光杆菌感染均有明显的免疫保护作用(表1)。
表1小鼠免疫保护实验
注：存活数为攻毒24小时后存活的小鼠数。
实施例4
大肠杆菌OmpA抗血清被动免疫增强作用：将纯化的大肠杆菌OmpA免疫家兔，获得抗血清，被动免疫鲫鱼(约30克/条)，以YfiO抗血清和生理盐水作为对照。每条注射50微升。同时用5倍半数致死量的相应细菌进行攻毒保护性实验，观察其死亡率。根据相对免疫保护率来比较分析这些免疫原性蛋白质的保护效果，结果发现对副溶血弧菌、铜绿假单胞菌或美人鱼发光杆菌感染均有明显的免疫保护作用(表2)。
表2大肠杆菌OmpA抗血清被动免疫保护作用
*P＜0.05-0.01
Claims (1)
1.大肠杆菌OmpA作为动物免疫调节剂的应用。
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| CN102539759A (zh) * | 2010-12-24 | 2012-07-04 | 光晟生物科技股份有限公司 | 符合个体化差异的免疫调节剂筛选方法 |
| CN102584992A (zh) * | 2011-01-06 | 2012-07-18 | 北京大北农科技集团股份有限公司 | 大肠杆菌外膜蛋白单克隆抗体及其制备方法和应用 |
| CN103911390A (zh) * | 2014-04-24 | 2014-07-09 | 江南大学 | 一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法 |
| WO2014183232A1 (zh) * | 2013-05-16 | 2014-11-20 | 河北科星药业有限公司 | 预防大肠杆菌病的基因tsh-Ser、其编码的蛋白质及其制备方法 |
| CN107523551A (zh) * | 2016-06-20 | 2017-12-29 | 中国农业科学院饲料研究所 | 一种抗大肠杆菌细胞株及重组单链抗体及其制备方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102539759A (zh) * | 2010-12-24 | 2012-07-04 | 光晟生物科技股份有限公司 | 符合个体化差异的免疫调节剂筛选方法 |
| CN102539759B (zh) * | 2010-12-24 | 2015-08-05 | 光晟生物科技股份有限公司 | 符合个体化差异的最适益生菌的筛选方法 |
| CN102584992A (zh) * | 2011-01-06 | 2012-07-18 | 北京大北农科技集团股份有限公司 | 大肠杆菌外膜蛋白单克隆抗体及其制备方法和应用 |
| CN102584992B (zh) * | 2011-01-06 | 2015-11-18 | 北京大北农科技集团股份有限公司 | 大肠杆菌外膜蛋白单克隆抗体及其制备方法和应用 |
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| CN103911390A (zh) * | 2014-04-24 | 2014-07-09 | 江南大学 | 一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法 |
| CN103911390B (zh) * | 2014-04-24 | 2017-02-15 | 江南大学 | 一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法 |
| CN107523551A (zh) * | 2016-06-20 | 2017-12-29 | 中国农业科学院饲料研究所 | 一种抗大肠杆菌细胞株及重组单链抗体及其制备方法 |
| CN110302369A (zh) * | 2019-06-28 | 2019-10-08 | 中国人民解放军陆军军医大学 | 以壳聚糖修饰PLGA的大肠杆菌Vo外膜蛋白纳米粒疫苗及其制备方法和应用 |
| CN110302369B (zh) * | 2019-06-28 | 2022-04-29 | 中国人民解放军陆军军医大学 | 以壳聚糖修饰PLGA的大肠杆菌Vo外膜蛋白纳米粒疫苗及其制备方法和应用 |
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