CN1092069C - 用于预防荚膜生物所致疾病的重组疫苗 - Google Patents
用于预防荚膜生物所致疾病的重组疫苗 Download PDFInfo
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Abstract
本发明涉及的疫苗由经过遗传修饰，即，使荚膜合成的编码基因或其一部分缺失而产生的无荚膜型突变微生物制备，用于预防常态下带有荚膜的微生物引起的疾病。作为实施例，一种编码荚膜合成的染色体DNA大片段缺失的活的减毒胸膜肺炎放线芽孢杆菌是预防猪胸膜肺炎的安全有效的疫苗。
Description
发明背景
发明领域
本发明涉及兽医应用的疫苗，特别是用于预防由荚膜微生物所引起的疾病的活的重组减毒疫苗，所述微生物的荚膜为毒性必要因素，但不是免疫防护所必需的。本发明具体涉及经遗传工程修饰使荚膜缺失了的重组工程疫苗。
现有技术
疫苗是通过引起人或动物的免疫反应抑制特定疾病的制品。用针对具体疾病的抗原处理患者，使患者的免疫系统产生大量的抗体。血液中存在的抗体能够使患者免于致病抗原的后继攻击。疫苗种类包括抗原的亚基或活的、杀灭的抗原本身。例如，脊髓灰质炎，通常称为“灰质炎”，可以用活的、减毒的口服灰质炎病毒疫苗预防，此种预防常用于儿童；或者使用杀灭的、失活的灰质炎病毒疫苗，此举经常用于成人，因为用活疫苗感染骨髓灰质炎危险性要高。如果使用活疫苗，必须以某种方式减毒；否则疫苗中的病毒会引起所要预防的疾病。
许多疾病由荚膜微生物引起，荚膜是这些微生物细胞壁外的胶状的多糖或多肽层，是致病的必要成分。猪胸膜肺炎为一例，引起该病的胸膜肺炎放线芽孢杆菌(Actinobacillus pleuropneumoniae)的毒性因子包括荚膜多糖、内毒素、蛋白外毒素。该病是世界范围内影响猪产量的一种主要的呼吸道疾病，仅在美国每年就造成上百万元的损失。
授权给Inzana的美国专利5,429,818，在此处引做参考，公开了一种胸膜肺炎放线芽孢杆菌的无荚膜型变异株没有毒性，并可提供对随后致病菌的攻击产生良好的抗性。Inzana描述的无荚膜型变异株是用甲基磺酸乙酯诱变得到的。然而，该方法的缺点在于某些自发或化学诱变的突变子不稳定，突变本质不清楚。
本发明概述
本发明的一个目的是提供一种用于预防由正常情况下带有荚膜的细菌或真菌引起的疾病的安全有效的活的减毒重组疫苗，所述荚膜是毒性而非免疫防护的必需成分。
本发明另一目的是用基因工程方法使细菌或真菌缺失荚膜，失去毒力，且弄清突变的遗传本质。
本发明再一目的是提供预防胸膜肺炎的安全有效的活的减毒重组疫苗。
根据本发明，通过基因工程获得一个缺失荚膜的重组胸膜肺炎放线芽孢杆菌的活性减毒株。由于荚膜为毒性而非免疫防护所必需，该菌株可用作抗猪胸膜肺炎的疫苗。该疫苗由不能在胸膜肺炎放线芽孢杆菌中复制的克隆质粒载体制备。第5血清型胸膜肺炎放线芽孢杆菌菌株的荚膜输出和合成基因已被测序。对克隆出的荚膜合成基因做大片段删除，在删除位点克隆插入卡那霉素抗性和蔗糖敏感基因作为标记基因。将这个自杀载体用电穿孔方法插入第5血清型胸膜肺炎放线芽孢杆菌菌株，以便在染色体和载体质粒的同源区发生双交换产生同源重组。得到失去彩虹色，表明缺失荚膜的四株重组菌。分别用点印迹和Southern印迹证实了每个菌株均缺失荚膜及有荚膜基因的删除区。同时证实重组菌株中存在标记基因。没有识别出任何其他表现型的变化，在染色体其他区域也没有发现标记基因。重组菌株，命名为J45-100，血清敏感性强，毒力减低，对猪的50％致死剂量为亲本的10倍，能够使猪对胸膜肺炎产生抗性。
本发明可用于制备抗产生毒素或其他毒性因子的任何有荚膜微生物的疫苗，其荚膜为毒性而非免疫防护所必需。所需工作是克隆该微生物的编码荚膜合成的编码基因，并在自杀载体上删除该基因并用标记基因代替该克隆基因，然后将载体引入理想的宿主，筛选缺失荚膜的经基因修饰的微生物。本发明可用于制备针对其他感染性细菌以及真菌如新型隐球酵母(Cryptococcus neoformans)的疫苗，所述细菌包括但不限于出血败血性巴斯德氏菌(Pasteurella multocida)、溶血性巴斯德氏菌(Pasteurella haemolytica)、铜绿假单胞菌(Pseudomonas aeruginosa)，所述真菌是与猫和人类获得性免疫缺陷综合症(AIDS)有关的致病原。
附图简述
下面结合附图对本发明优选实施方案进行详述，这将有助理解前述及其他的本发明目的、方面和优点：
图1是带有胸膜肺炎放线芽孢杆菌J45的荚膜合成DNA片段的克隆载体pCW-11E的物理图谱。图中示出了以双脱氧测序确定的两个完整的ORFs(cpsA和cpsB)的转录位置和方向。并示出了第三个潜在ORF(cpsC)的部分片段的位置及不完整的荚膜输出基因cpxD在该DNA片段上的位置和转录方向。图2中用作DNA探针的2.1Kb的Bg/ll-Stul片段也有显示。打点的箭头表示不完整的ORFs。
图2是胸膜肺炎放线芽孢杆菌基因组DNA与异羟基洋地黄毒苷配基标记的pCW-11E的2.1Kb长的Bg/ll-Stul片段杂交的Southern印迹。来自第1血清型菌株4074(第1道)、第2血清型菌株1536(第2道)、第5a血清型菌株J45(第3道)、第5a血清型菌株K17(第4道)、第5血清型菌株178(第5道)、第7血清型菌株29628(第6道)和第9血清型菌株13261(第7道)的基因组DNA经BamH1消化后如下与探针杂交。图中示出了杂交条带的分子量大小(kb)。
图3a和3b表示pCW-11E的3.2kb长的HindIII-EcoRV片段的核苷酸序列，该片段包含胸膜肺炎放线芽孢杆菌J45血清型特异的DNA(SEQ ID NO.1)。据序列中两个完整ORFs，cpsA(SEQ ID NO.2)和cpsB(SEQ ID NO.3)推测出的氨基酸序列、据第三个不完整ORF，cpsC(SEQ ID NO.4)推测出的N-末端序列示于核苷酸序列之下。推定的位于各ORF前的核糖体结合位点以粗体字表示，并示出了推定的位于cpsA上游-10和-35处的启动子序列。
图4描述含有缺失荚膜合成DNA的自杀载体pCW11EΔ1KS1的构建，由等位基因交换产生胸膜肺炎放线芽孢杆菌J45的无荚膜型突变子。用Bg/ll和Stul消化pCW-11E，形成平末端，将所得的6.4kb大片段与3.8kb的含有npt1-sacRB(Kanr Sucs)的pKS的BamHI片段(也是平末端)连接，由此构建pCW11EΔ1KS1质粒载体。括号内的限制酶切位点表示在pCW11EΔ1KS1中连接的片段的初始末端。将载体pCW11EΔ1KS1载体电转化入胸膜肺炎放线芽孢杆菌，在含有85ug/ml卡那霉素的培养基上筛选缺失彩虹色的无荚膜型卡那霉素抗性转化子。
图5是自胸膜肺炎放线芽孢杆菌J45(第1道)或J45-100(第2道)分离出的基因组DNA与异羟基洋地黄毒苷配基标记的特异地识别npt1或胸膜肺炎放线芽孢杆菌荚膜形成座位的探针的Southern印迹。胸膜肺炎放线芽孢杆菌J45(第1道)或J45-100(第2道)的基因组DNA经Xba1消化(A列和C列)或BamH1消化(B列)，与pKS的1.24kb的Pst1(npt1特异的)片段杂交，A列；或与pCW-11E的2.1kb的Bg/ll-Stul片段(cpsABC特异的，参见图1)杂交，B列；或与pCW-1C的2.1kb的c/al片段(cpxCBA特异的，参见图3.2)杂交，C列。
图6是胸膜肺炎放线芽孢杆菌J45或J45-100与荚膜多糖特异的猪抗血清反应形成的菌落免疫印迹。每孔约5×105(第1道)或5×104(第2道)CFU转至硝酸纤维素膜，以氯仿处理该硝酸纤维素膜，并使之与猪抗血清温育，该抗血清含有第5a血清型荚膜多糖的抗体，而不含针对胸膜肺炎放线芽孢杆菌其他表面抗原的抗体。
图7显示胸膜肺炎放线芽孢杆菌J45(第1道)和J45-100(第2道)的主要含外毒素Apx1和Apx11的浓缩培养物上清液的免疫印迹。A列与Apx1特异的单克隆抗体反应，B列和Apx11特异的单克隆抗体反应。A列中第2血清型胸膜肺炎放线芽孢杆菌菌株1536(第3道)的浓缩培养物上清液作为阴性对照，因为该血清型不合成Apx1。A列中的印迹与Apx1特异的单克隆抗体反应。
图8显示自胸膜肺炎放线芽孢杆菌J45(第1道)和重组无荚膜型突变子J45-100(第2道)分离出的LPS的电泳图谱。LPS用15％分离胶电泳，以银氨盐染色。
图9显示预乳化的小牛血清对胸膜肺炎放线芽孢杆菌J45和J45-100的杀菌活性。37℃温育60分钟后测定菌株的存活率。每个数据点代表三次实验的均值，每次实验设两个重复。误差线表示每个均值的标准误差。J45最大存活率记作100％，尽管数值一般因为细菌在实验过程中生长而偏高。大于100％的数值未记录，因为不能精确定值。
图10a和10b列出pCW-1C中编码胸膜肺炎放线芽孢杆菌J45荚膜输出基因的3.2Kb的Xba1-ClaI片段的核苷酸序列(SEQ ID NO.5)。图中示出了推定的参与输出第5a血清型胸膜肺炎放线芽孢杆菌荚膜多糖的蛋白的氨基酸序列(SEQ ID NO.6-8)。
图11是来自胸膜肺炎放线芽孢杆菌J45的pCW-1C DNA的物理图谱。
发明优选实施方案详述
本发明试图将通过基因工程获得的活的无荚膜型重组减毒微生物(即细菌或真菌)菌株作为疫苗，用于抗该微生物引起的疾病。本发明可用于预防由荚膜是毒性而非免疫防护的必需成分的微生物引起的疾病或由毒素或其他毒性因子引起的疾病。作为本发明一个具体的实施例，制备了无荚膜型的胸膜肺炎放线芽孢杆菌，可用作预防猪胸膜肺炎的疫苗。本发明主要特点在于对微生物，在特定实施方案中是对胸膜肺炎放线芽孢杆菌的基因进行修饰，包括删除荚膜合成编码区DNA。仅作为例子，本文公开了转化的第5a血清型胸膜肺炎放线芽孢杆菌突变子的制备方法，应理解的是：其他血清型突变子可以与下文描述的相似的方法制得，并以一种重组突变子或不同血清型的多种突变子联合作为疫苗。
下述菌株，以及其他按照下面的方法制备的产毒细菌或其他微生物的无荚膜型菌株，是非常好的疫苗，因为它们无毒，但能给宿主提供产生保护性免疫反应的所有必要抗原。疫苗有多种施用方法；优选肌内和皮下注射。这些活疫苗的优点在于作为致病主要原因的毒素和其他仅由活菌体或在体内才产生的成分在免疫位点产生，宿主对其产生免疫反应，使自身免于毒素的侵害，因此不会发病(急性或慢性)。所述微生物不能播散，但因为没有荚膜，其对血清高度敏感，在血液或呼吸道内将被立即清除。另外，活疫苗比死疫苗的细胞免疫反应强，保护力更持久。
实施例
鉴定并特征分析涉及胸膜肺炎放线芽孢杆菌荚膜多糖生物合成的DNA区域。特异于参与第5a血清型胸膜肺炎放线芽孢杆菌J45荚膜多糖输出的cpxD基因的特异探针用来确定并克隆相邻的5.8kb的BamHIJ45基因组DNA片段。Southern印迹显示这一区域含有血清型特异的DNA。DNA序列分析显示这一区域含有两个完整开放阅读框：cpsA和cpsB，和一个不完整的潜在的第三开放阅读框cpsC。cpsA和cpsB均与参与大肠杆菌脂多糖和b型流感嗜血杆菌(Haemophilus influenzae)荚膜多糖生物合成的糖基转移酶有低的同源性。构建一个2.1kb的跨越克隆的cpsABC开放阅读框的删除并通过等位基因交换整合到J45染色体中以获得突变子J45-100。该突变子不产生胞内或胞外荚膜多糖，表明cpsA、cpsB和/或cpsC参与胸膜肺炎放线芽孢杆菌荚膜多糖的生物合成。J45-100的Apx毒素和脂多糖与有荚膜亲本菌株J45的相同。但J45-100比J45体外生长迅速。J45-100易被预乳化的小牛血清灭活，J45则不。J45-100毒性衰减，给猪吸入时，50％致死剂量是J45的三倍。当达到J45的50％致死剂量的六倍时，J45-100引起中度肺损伤，不会致死。这些结果显示荚膜多糖是胸膜肺炎放线芽孢杆菌血清抗性和毒力的主要决定因素。材料与方法
菌株，质粒和生长条件。
本研究所用菌株、质粒的描述见表1。提取基因组DNA和进行细菌学分析时，将胸膜肺炎放线芽孢杆菌在含有5ug/ml烟酰氨腺嘌呤二核苷酸(NAD)(Sigma Chemical Co.，St.Louis，Mo)的脑心浸出液(DifcoLaboratories，Detroit，Mich.)中，37℃振荡培养。作电穿孔时，将胸膜肺炎放线芽孢杆菌在含0.6％酵母提取物(Difco Laboratories)和5ug/mlNAD(TSY-N)的胰蛋白大豆培养液(Difco Laboratories)中，37℃振荡培养。作攻击耐受实验时，将胸膜肺炎放线芽孢杆菌在含5ug/ml NAD的Columbia培养液(Difco Laboratories)中37℃振荡培养。大肠杆菌在Luria-Bertani培养液(Sambrook等，1989)中常规培养，或用Terrific培养液(Tartof Hobbes，1987)培养以提取质粒。培养基中加入如下浓度的抗生素来保持大肠杆菌的质粒：氨苄青霉素(Amp)100ug/ml，卡那霉素(Kan)50ug/ml。筛选胸膜肺炎芽孢杆菌重组突变子时用85ug/ml的卡那霉素。
表1
| 所用菌株和质粒 | |
| 胸膜肺炎放线芽孢杆菌4074 | 第1血清型；(ATCC27088) |
| ATCCa 1536 | 第2血清型；(ATCC27089) |
| ATCCa J45 | 第5a血清型 |
| Fenwick等，1986a K17 | 第5a血清型 |
| Nielsen，1986a 178 | 第5血清型 |
| M.Mulks 29628 | 第7血清型 |
| L.Hoffman 13261 | 第9血清型 |
| J.Nicolet J45-C | 甲基磺酸乙酯诱变后分离出的无荚膜型突变子 |
| J45 Inzana等，1993aJ45-100来源的 | 来自J45菌株的重组无荚膜型突变子 |
| 大肠杆菌菌株XL1-Blue | recA1 endA1 gyrA96 thi-l hsdR17 supE44relA1 lac(F+proAB laclqZΔM15 Tn10)；重组质粒的宿主 |
| Stratagene，La Jolla，Calif.质粒pGEM-3Z | 克隆载体，2.74kb；Ampr |
| Promega pCW-1C | 克隆至pGEM-3Z的J45的5.3kb Xba1片段 |
| PCW-11E | 克隆至pGEM-3Z的J45的5.8kb BamH1片段 |
| PKS | 克隆至pGEM-3Z的BamH1位点的3.8kb含有npt1b-sacRB“药筒”c的pKSBamH1片段；Ampr，Kanr |
| S.M.Boyle pCW11EΔ1KS1 | 缺失2.1kb Bg/ll-Stul片段的pCW-11E和来自pKS的3.8kb的npt1b-sacRB“药筒”c |
| a美国典型培养物保藏中心，Rockville，MD | |
| b该标记来源于pUC4K(Pharmacia Biotech，Piscataway，NJ)的Tn903 npt1基因 | |
| c“药筒”特性已知(Ried和Collmer，1987) | |
增代时间的计算。生长于TSY-N的胸膜肺炎放线芽孢杆菌对数期用下列公式计算：R＝l/g，其中R是细菌平均生长速率，g是菌群的增代时间(Pelczar等，1993)。平均生长速率R用下列公式计算：R＝3.32(lgN-lgN0)/t，其中t是培养时间，N是t时刻的细菌数目，N0是0时刻的细菌初始数目(Pelczar等，1993)。
DNA杂交分析。经限制性内切酶消化的DNA(每道约5ug)用0.7％琼脂糖凝胶电泳，按照以前的方法(Sambrook等，1989；Southern，1975)采用20X醋酸钠盐水溶液(20X醋酸钠盐水溶液是3M NaCl，300mM醋酸钠，pH7)利用毛吸作用转移到MagnaGraph尼龙膜(MicronSeparations Inc.Westboro.Mass)上。用一个紫外Stratalinker(Stratagene，LaJolla，Calif.)进行紫外照射使DNA与膜共价联接。DNA杂交所用异羟基洋地黄毒苷配基标记的探针以随意引物方法用Genius System非放射性标记和检测试剂盒(BoehringerMannheim Corp.，Indianapolis，Ind.)合成，合成过程参见厂商说明。DNA杂交在含5X SSC的溶液中，于68℃进行。按照Genius System使用说明清洗膜并显影进行比色检测。
DNA重组方法和试剂。基因组DNA用S.Spinola描述的方法自肉汤培养的胸膜肺炎放线芽孢杆菌中分离。简要步骤是，将细菌悬浮于10mM Tris--1mM EDTA(pH8)中，与十二烷基磺酸钠(0.66％)共育，并用RNAse(100ug/ml)37℃处理1小时。加入蛋白酶K至终浓度为100ug/ml，使混合液于56℃温育1小时。所得混合液用缓冲的酚提取一次，再用缓冲的酚-氯仿(Amresco，Inc.，Solon，Ohio)提取四次，基因组DNA用乙醇沉淀，并重悬于10mM Tris--1mM EDTA(pH8)中。从快速碱裂解法(Ish-Horowicz和Burke，1981)分离质粒DNA。将克隆和合成探针所需的限制酶切片段从琼脂糖凝胶洗脱出来(Zhen Swank，1993)。限制酶切、琼脂糖凝胶电泳和DNA连接按前述方法(Sambrook等，1989)进行。限制酶切片段的末端用DNA聚合酶1的Klenow片段在5’添加核苷酸(dNTPs)使成平末端，方法见前述(Sambrook等，1989)。质粒DNA用BTX ECM600电穿孔仪(BTX，Inc.，San Diego，Calif.)通过电穿孔转化入大肠杆菌(Dower，1988)。
限制性内切酶和DNA聚合酶I的Klenow片段购自Promega公司(Madison，Wis.)。T4 DNA连接酶购自Gibco BRL公司(Gaithersburg，Md.)。填平反应所用核苷酸购自Boehringer-Mannheim公司(Indianapolis，Ind.)。
DNA测序和分析。pCW-11E的2.7kb的Xbal-EcoRV DNA片段的双链核苷酸序列采用35[S]dATP(DuPont/NEN Research Products，Boston，Mass.)用Sequenase version 2.0 DNA测序试剂盒(美国生化公司，Cleveland，Ohio)以双脱氧链终止方法(Sanger，1977)确定。用寡核苷酸引物(DNAgency，Inc.，Malverne，Pa.)沿每链顺序阅读测定双链DNA模板序列。
将测得的核苷酸序列与pCW-1C中编码荚膜结构基因上游序列的4.6kb Xbal-Clal DNA片段的核苷酸序列(图10)结合，并用DNASTAR软件(DNASTAR，Inc，Madison，Wis)进行分析。序列相似性用BLAST软件(Altschul，1990)检索国家生物技术信息中心(Bethesda，Md.)的EMBUGenBank/DDBJ数据库。
用与b型流感嗜血杆菌荚膜多糖输出有关的cap(capb)基因座位的保守区确定、克隆和定性参与第5a血清型胸膜肺炎放线芽孢杆菌荚膜多糖输出的荚膜形成座位的一部分。用b型流感嗜血杆菌capb位点的特异探针做Southern印迹，分析第5a血清型胸膜肺炎放线芽孢杆菌菌株J45基因组DNA。这些探针在高严谨条件下(68℃，5X SSC)不与胸膜肺炎放线芽孢杆菌基因组DNA杂交，但在中到低度严谨条件下(55℃，5X SSC)却与胸膜肺炎芽孢杆菌的基因组DNA发生杂交。来自质粒pSKH1的b型流感嗜血杆菌capb基因的一个4.4kb EcoRI片段含有与荚膜多糖输出有关的区域1 bexD基因和参与荚膜生物合成的两个区域2开放阅读框(ORFs)，该片段与J45基因组DNA的1.2kb HindIII片段和5.3kb XbaI片段杂交。来自质粒pSKH 2的b型流感嗜血杆菌capb座位的9.0kb EcoRI片段含有与荚膜多糖输出有关的区域1 bexCBA基因、流感嗜血杆菌某些血清型共有的一些特征不明的区域3 DNA以及参与荚膜多糖的生物合成的几个区域2 DNA，该9.0kb片段与J45基因组DNA的1.5kb HindIII片段、5.3kb XbaI片段和2.4kb XhoI片段杂交。这些结果表明b型流感嗜血杆菌与第5a血清型胸膜肺炎放线芽孢杆菌的荚膜基因座位有同源区。b型流感嗜血杆菌capb座位的特异探针都含有与荚膜多糖输出有关的区域1 DNA，提示与b型流感嗜血杆菌capb探针杂交的来自J45基因组DNA的5.3kb XbaI片段可能含有编码参与第5a血清型胸膜肺炎芽孢杆菌荚膜多糖输出的蛋白的编码基因。将上述5.3kb来自J45基因组的XbaI片段(以两种方向)克隆至质粒pGEW-3Z的XbaI位点，质粒pGEW-3Z来自XbaI消化J45基因组DNA，琼脂糖凝胶电泳后洗脱得到的4.8到6.0kb片段。制备的质粒之一命名为pCW-1C。做Southern印迹确定b型流感嗜血杆菌的bexD，bexC，bexB和bexA与pCW-1C的相邻片段杂交顺序是否与这些基因在b型流感嗜血杆菌中的顺序(bex DCBA)相同。结果提示第5a血清型胸膜肺炎放线芽孢杆菌荚膜多糖输出必需的DNA区域已被成功地克隆，且在该区域基因组织方式与b型流感嗜血杆菌中的bex座位相似。
测定pCW-1C的4.6kb XbaI-ClaI限制酶切片段的核苷酸序列，图10a-b显示出3.2kb XbaI-ClaI限制酶切片段的核苷酸序列(SEQ IDNO.5)。在同一DNA链上检测到紧邻的四个命名为cpxDCBA(cpx代表荚膜多糖输出)的ORF(图10a-b和图11示出)。cpxC(SEQ ID NO.7)的起始密码子AUG在cpxD(SEQ ID NO.6)终止密码子UAA下游26个核苷酸处，而cpxB(SEQ ID NO.8)的AUG起始密码子与cpxC终止密码子UAA重叠，cpxA的起始密码子AUG与部分存在的cpxB(SEQ IDNO.8)终止密码子UGA重叠。在每个起始密码子AUG上游17个碱基范围内都有核糖体结合通用序列Shine-Dalgamo，在cpxD(SEQ ID NO.6)上游鉴定出一个推定的启动子，该启动子含有与大肠杆菌670-10(TATAAT)和-35(TTGACA)通用序列相似的序列。在cpxA(未图示)下游鉴定出一个可能具有rho-非依赖型转录终止信号功能的回文序列。上述的基因排布方式表明cpxDCBA被转录于一个多顺反子mRNA。
胸膜肺炎放线芽孢杆菌的电转化。胸膜肺炎放线芽孢杆菌在TSY-N培养基中生长至对数中期，于4℃以7000g离心沉淀，用冷的(4℃)经过过滤除菌的含有272mM甘露醇、2.43mM K2HPO4、0.57mM KH2PO4、15％甘油，pH7.5的缓冲液洗四次，该缓冲液是经过改进的以前用于电穿孔前洗涤胸膜肺炎放线芽孢杆菌细胞的缓冲液(Lalonde等，1989b)。然后用冷的过滤除菌的15％甘油再洗一次上述的细胞，以约1010CFU/ml将之悬浮于15％甘油。悬液(90ul)与氯化铯密度梯度超离心提纯的1.5-2.0ug质粒DNA(在1.5ml蒸馏水中)混合，放置在冷的2mm间隔的电穿孔管(BTX，Inc.)中，用BTXECM600电穿孔仪(BTX，Inc.)将充电压调至2.5KV，电阻用R7档(246 ohms)进行电穿孔。实际产生的电脉冲是10.7毫秒内输出电压2.39KV。电穿孔后，细胞在含有5mMMgCl2的1ml TSY-N中，于37℃轻微振荡3.5小时后回收。然后用每ml含85ug卡那霉素的TSY-N琼脂培养基，37℃培养所得细胞。
免疫印迹。为了做菌落免疫印迹，将经TSY-N琼脂平板过夜培养的胸膜肺炎放线芽孢杆菌刮到磷酸盐缓冲液(PBS)中，根据比色调至109CFU/ml。用Bio-Dot器(Bio-Rad laboratories，Richmond，Calif.)以每孔约5×104或5×105CFU转至硝酸纤维素膜(NitroBind，Micron SeparationsInc.)。将所述膜于室温放入氯仿15分钟，裂解膜上的菌体，使膜完全风干后，放入pH7.5含2％脱脂牛奶的Tris缓冲盐水溶液(TBS)中，于室温温育1小时，以此来封闭膜上非特异性结合位点。使膜在1∶200稀释度(在2％牛奶-TBS中)的经吸附的猪抗血清中于室温保持1小时，该抗血清中含有抗第5a血清型荚膜多糖的抗体，而无抗胸膜肺炎放线芽孢杆菌其他表面抗原的抗体。这种富集荚膜多糖的抗血清的制备按照前述方法(Inzana，1995)，用自发无荚膜型突变株K17-C(Inzana和Mathison，1987)以超免疫猪抗血清吸附胸膜肺炎放线芽孢杆菌K17。上述膜用含0.05％Tween20的TBS洗涤，于室温在1∶1000稀释度的联结辣根过氧化酶的兔抗猪IgG(重链和轻链；Cappel，Durham，N.C.)中保温1小时。膜用TBS洗后，然后用4-氯-1-萘酚(Bio-Rad Laboratories)在含0.02％H2O2的TBS中显影。
胸膜肺炎放线芽孢杆菌的浓缩培养物上清液的免疫印迹按照前述方法(Ma和Inzana，1990)进行。简言之，用不连续SDS-PAGE(Laemmli，1970)通过8％分离胶分离得到约上清液中的总蛋白15ug。按照Towbin等的方法(1979)，将蛋白转至硝酸纤维素膜(NitroBind；Micron Separations Inc.)。将膜在含2％牛血清白蛋白的TBS中保温，封闭非特异结合，将膜切成条，于4℃使切成条的膜或者与Apx11毒素的特异单抗(Ma和Inzana，1990)或与Apx1毒素的特异单抗(Devendish等，1989；Frey等，1992)保温过夜，然后在TBS中洗。使与Apx11特异单抗反应的印迹和1∶2000稀释度的联结辣根过氧化酶(cappel)的羊抗鼠IgG保温，在TBS中洗后，按前面的方法显影。使与Apx1特异单抗反应的印迹和1∶2000稀释度的联结碱性磷酸酶的羊抗鼠IgG保温，按前面的方法显影(Frey等，1992)。
LPS的提取和电泳。LPS用微量热苯酚-水提取法(Inzana，1983)从胸膜肺炎放线芽孢杆菌分离。纯化的LPS在含脲的15％聚丙烯酰胺分离胶中电泳(Inzana，1988)，LPS电泳图谱用银铵染色显形(Tsai和Frasch，1982)。
血清杀菌分析。检测胸膜肺炎放线芽孢杆菌对预乳化的小牛血清杀菌力的敏感度。于37℃使菌株分别与5、10、15、20、30、40、50％预乳化的小牛血清温育60分钟后，测定细菌的存活率。
毒性研究。7-9周龄未感染胸膜肺炎放线芽孢杆菌的猪来自两个当地畜群，随机分组，各组猪分开饲养，各组间无直接接触。实验动物在弗吉尼亚工学院和州立大学的饲养和操作按照美国实验室动物保护法协会的要求进行。作攻击耐受实验时，胸膜肺炎放线芽孢杆菌在补充有5ug/ml NAD的Columbia肉汤(Difco laboratories)中振荡培养，以7000g离心，以约109CFU/ml重新悬浮于PBS中。猪用Stresnil(Pittman-Moore，Inc.Washington Crossing，N J.)镇静后，经气管摄入10ml稀释的上述悬液。在猪死后或用戊巴比妥钠实施安乐死后立即解剖，由兽医病理学家按下列标准对肺损伤进行评分：0，不显著肺(无可见损伤)；1+，1-10％肺组织充血、水肿、出血、实变，和/或胸膜炎；2+，11-49％肺组织病变；3+，50-74％肺组织病变；4+，75％或以上肺组织病变。肺样品取自尾叶的右颅背侧，在含NAD的脑心浸出液中培养以检测是否存在胸膜肺炎放线芽孢杆菌。结果
胸膜肺炎放线芽孢杆菌血清型特异DNA区的鉴定与克隆。为了鉴定和克隆胸膜肺炎放线芽孢杆菌J45中与荚膜生物合成有关的DNA，先作Southern印迹，以便鉴定cpxDCBA上游(5’方向)相邻的DNA区，cpxDCBA基因簇如前文所述参与荚膜多糖的输出(图10a-b和11)。预计这个上游DNA区编码血清型特异的荚膜多糖生物合成基因，因为胸膜肺炎放线芽孢杆菌荚膜(cap)基因座位的组织排布方式似乎与b型流感嗜血杆菌和脑膜炎萘瑟氏球菌(Neisseria meningitidis)B族中的相似。用异羟基洋地黄毒苷配基标记的包含部分cpxD基因的pCW-1C的1.2kbBamHI-Xba1片段作为探针，来检测BamHI消化的胸膜肺炎放线芽孢杆菌J45基因组DNA。该cpxD特异的探针与J45基因组DNA一个约5.8kb的BamHI片段杂交(结果未显示)。将上述5.8kb BamHI片段克隆到pGEW-3Z的BamHI位点，所述pGEM-3Z来自J45基因组DNA经BamHI消化并经电泳分离后自琼脂糖凝胶电洗脱得到的5.0-6.5kb片段。形成的质粒命名为pCW-11E，测定其限制酶切图谱(图1)。部分pCW-11EDNA插入片段(1.2kb BamHI-Xba1片段)与pCW-1C的插入片段重叠。
来自不同血清型胸膜肺炎放线芽孢杆菌基因组DNA的BamHI消化片段用pCW-11E的2.1kb Bg/ll-Stul片段杂交(图1)，鉴定这个DNA区域的血清型特异性(图2)。该2.1kb的Bg/ll-Stul片段与来自所检测的3个第5血清型胸膜肺炎放线芽孢杆菌菌株的5.8kb BamHI片段杂交，而不与第1、2、7和9血清型菌株的基因组DNA杂交(图2)。因此，pCW-11E中的胸膜肺炎放线芽孢杆菌DNA含有第5血清型菌株的特异性DNA。由于这个DNA是血清型特异性的，它可能与荚膜多糖生物合成有关。
胸膜肺炎放线芽孢杆菌血清型特异DNA区域的核苷酸序列与分析。对pCW-11E的2.7kb Xbal-EcoRV DNA片段核苷酸序列进行测定。测得的核苷酸序列结合pCW-1C的4.6kb Clal-Xba1 DNA片段的核苷酸序列，检查是否有以前未被识别的开放阅读框(ORFs)。含新鉴定的ORFs的pCW-11E的3.2kb HindIII-EcoRV片段见图3。两个分别命名为cpsA和cpsB(cps表示荚膜多糖生物合成)的完整ORFs，在互补链上位于胸膜肺炎放线芽孢杆菌荚膜多糖输出相关基因cpxD的上游(图1和图3)。cpsB起始密码子AUG在cpsA终止密码子UAA下游3个核苷酸处。第三个潜在ORF cpsC起始密码子AUG在cpsB终止密码子UAA下游15个碱基处。在cpsA、cpsB、cpsC起始密码子AUG上游13个碱基范围内都有核糖体结合通用序列Shine-Dalgarno(Shine和Dalgarno，1974)(图3)。在cpsA上游(图3)鉴别出一个推定的启动子，该序列含有与大肠杆菌670的-10(TATAAT)和-35(TTGACA)通用序列(Hawley和McClure，1983)相似的序列。cpsABC的紧密邻接以及推定启动子上游区的鉴定提示这些ORF可能是共转录的。编码cpsABC的DNA区，其G+C含量为28％。
推测cpsA和cpsB的多肽分别含有321(cpsA)和526(cpsB)个氨基酸(图3)。CpsA和CpsB推测分子量分别为36.9和61.7Kda。亲水曲线表明CpsA和CpsB是相对亲水的蛋白，提示它们可能与胞质结构有关联(数据未显示)。用BLAST(Altschul等，1990)检索国家生物技术信息中心(Bethesda，Md.)的结合、非冗余核苷酸和蛋白数据库，显示CpsABC在核苷酸和氨基酸水平与数据库其他序列无基本同源性(数据未显示)。但CpsA与大肠杆菌中参与LPS生物合成的O-抗原糖基转移酶Rfb蛋白(Cheah和Manning，1993)有低的同源性(15％相似)，CpsB与b型流感嗜血杆菌荚膜基因座位的区域2的ORF3蛋白产物有低的同源性(约14％相似)。ORF3蛋白参与b型流感嗜血杆菌多核糖的核苷磷酸荚膜多糖生物合成(Van Eldere等，1995)。CpsC N端的83个氨基酸与数据库中的任何蛋白无明显同源性。
卡那霉素抗性无荚膜型胸膜肺炎放线芽孢杆菌转化子的制备。图4概括了通过同源重组和等位基因交换制备无荚膜型胸膜肺炎放线芽孢杆菌J45突变子的步骤。首先构建pCW11EΔ1KS1作为非复制型自杀载体以促进野生型与基因发生了改变的胸膜肺炎放线芽孢杆菌DNA间的双同源重组交换。构建载体pCW11EΔ1KS1时，首先用Bg/ll和Stul消化pCW-11E，在胸膜肺炎放线芽孢杆菌血清型特异荚膜形成DNA中缺失大片段。将该消化得到的DNA片段补平为平末端，将此6.4kb大片段连接3.8kb的含有npt1-sacR-sacB“药筒”的pKS的BamHI片段(也是平末端)。“药筒”含有已知的Tn903 npt1基因和sacRB序列，前者使胸膜肺炎放线芽孢杆菌具备卡那霉素抗性(Kanr)(Tascon等，1994)，后者赋予多种革兰氏阴性菌以蔗糖敏感性(Sucs)(Gay等，1983；Ried和Collmer，1987)。所述在pCW11EΔ1KS1上产生的缺失片段跨越cpsABC(图1、图4)，因此可能影响这些蛋白产量。
pCW11EΔ1KS1载体在胸膜肺炎放线芽孢杆菌中不复制，可作为自杀载体。电穿孔使pCW11EΔ1KS1进入胸膜肺炎放线芽孢杆菌J45后，回收培养体系在37℃培养两天后得到7个卡那霉素抗性转化子，这些卡那霉素抗性J45转化子中的四个在斜射光源下无虹彩，提示是无荚膜型(数据未显示)。pCW11EΔ1KS1电穿孔前，胸膜肺炎放线芽孢杆菌的培养基是一个影响因素，因为使用补充有NAD的脑心培养胸膜肺炎芽孢杆菌浸液时，从未得到过抗性转化子。
卡那霉素抗性胸膜肺炎放线芽孢杆菌转化子的基因型特征。七个卡那霉素抗性转化子菌落的预杂交分析显示：没有荚膜(目测)的四个转化子与npt1特异的DNA探针(pKS的1.24kb Pst1片段)杂交，但不与pGEW-3Z的特异探针(pGEW-3Z的1.1kb Bg/ll片段)或血清型特异的pCW-11E的2.1kb Bg/ll-Stul片段杂交。这些结果表明四个卡那霉素抗性转化子均发生了双交换重组。相反，其他三个卡那霉素抗性转化子菌落与npt1基因、pGEW-3Z、pCW-11E 2.1kb Bg/ll-Stul片段特异的探针杂交，表明仅发生了单交换，完整的pCW11E1ΔKS1自杀载体已整合到这些转化子的染色体中(数据未显示)。从四个卡那霉素抗性、潜在的无荚膜型转化子提纯的基因组DNA其Southern印迹(采用如上所述的探针)结果相同，表明每个转化子发生了同样的双交换重组。随机挑选一个转化子进行进一步研究并命名为J45-100。
对从J45、J45-100分离出的基因组DNA和特异识别npt1基因的DNA探针、pCW-11E的2.1kb Bg/ll-Stul片段和pCW-1C的2.1kb Clal片段进行Southern印迹分析(图5)。npt1特异性DNA探针与J45-100经Xba1消化产生的5.0kb片段杂交，而不与J45 DNA杂交，验证了npt1标基因存在于J45-100基因组中(图5A)。npt1探针杂交的J45-100的5.0kb Xba1片段与制备J45-100所用的pCW11E1ΔKS1自杀载体内的Xba1片段大小一致。pCW-11E的2.1kb Bg/ll-Stul片段与J45的5.8kbBamHI片段杂交，而不与J45-100的DNA杂交，验证了这个片段在J45-100中被缺失(图5B)。与荚膜多糖输出有关的cpxCBA基因(pCW-1C的2.1kb Clal片段)的特异探针与J45和J45-100的5.3kbXba1片段都杂交(图5C)，这一结果验证了胸膜肺炎放线芽孢杆菌荚膜形成基因座位的这一部分，不受邻近区域发生的双交换重组的影响。pGEW-3Z的特异探针与J45和J45-100的基因组DNA都不杂交，验证了J45-100的基因组不含有载体DNA。总而言之，这些杂交结果表明J45-100中发生了所需的双交换重组和等位基因交换。
卡那霉素抗性胸膜肺炎放线芽孢杆菌转化子J45-100的表现型特征。J45-100荚膜多糖产量用菌落免疫印迹和乳胶凝集试验测定。含有特异结合第5血清型胸膜肺炎放线芽孢杆菌荚膜多糖的抗体但不含其他表面抗原抗体的抗血清与J45反应，但不与J45-100反应(图6)。因为膜上的菌落已用氯仿裂解，这些结果表明J45-100不产生胞外或胞内荚膜多糖。完整或超声破碎的J45-100不与联接了第5血清型荚膜多糖的纯化抗体(Inzana，1995)的乳胶珠凝集，而J45完整细胞和超声破碎的J45-C细胞使乳胶珠发生强凝集反应(数据未显示)。上述结果验证了胸膜肺炎放线芽孢杆菌J45-100cap基因座位中的缺失造成荚膜多糖生物合成能力丧失。结果还表明甲基磺酸乙酯诱变J45后得到的无荚膜型突变子J45-C(Inzana，1993a)产生胞内但不产生胞外荚膜多糖。
比较J45和J45-100 Apx毒素的表达和LPS电泳图谱，确定J45-100的cap基因座位中的突变是否影响这些重要的毒性决定因素。J45和J45-100培养基上清液中105kDa的Apx1和Apx11分泌量没有不同(图7)，另外，两者的LPS电泳图谱也没有差别(图8)。
检测J45和J45-100在TSY-N培养液的生长情况以及它们对预乳化的小牛血清杀菌力的敏感性，确定失去荚膜合成能力对表现型的影响。J45和J45-100在TSY-N中的生长曲线相似，但不完全相同(数据未显示)。但是平板活菌计数显示在对数生长期，J45-100(增代时间＝ca.23分钟)比亲本荚膜型菌株J45(增代时间＝ca.28分钟)生长更快(数据未显示)。重组无荚膜型突变子J45-100在10-50％预乳化的小牛血清作为补体时在60分钟内被有效地杀灭，而荚膜型亲本菌株J45却不被杀灭(图9)。
检测J45-100对蔗糖的敏感性以确定sacRB是否可作为胸膜肺炎放线芽孢杆菌反选标记并随后诱导从J45-100染色体删除npt1-sacRB抗药片段。肉汤培养的J45-100在直接或稀释后铺含有5％或8％蔗糖的TSY-N或luria-Bertani(加5ll/ml NAD)平板后，其生长旺盛。Southern印迹验证J45-100染色体中存在sacRB序列。这些结果提示sacRB标记或者在胸膜肺炎放线芽孢杆菌不表达，或者可能由sacRB果聚糖蔗糖酶在蔗糖存在下生成的果聚糖对J45-100无毒性。
猪气管摄入重组无荚膜型胸膜肺炎放线芽孢杆菌突变子J45-100对猪进行攻击。在施用重组无荚膜型突变子J45-100的量是荚膜型亲本J4550％致死剂量(LD50)(5×106 CFU)(Inzana，1993a)的3到6倍(分别是1.45×107CFU和×107)时，不引起猪的死亡(表2)。在给药量是J45 LD50的6.5倍时，三头受攻击的猪均形成严重的肺损伤而死亡(表2)。
表2
| 胸膜肺炎放线芽孢杆菌J45和J45-100对猪的毒性 | ||||
| 阳性/实验总数 | ||||
| 攻击菌株 | 攻击剂量 | 平均肺损伤度 | 死亡率 | 回收a |
| J45 | 1.6-3.30×107CFUb | 4+ | 3/4c | 4/4 |
| J45-100 | 1.5×107CFU | 0 | 0/5 | 0/5 |
| J45-100 | 3.0×107CFU | 1+ | 0/5 | 2/5d |
| J45-100 | 8.4×107CFU | 1+ | 1/4e | 4/4d |
| J45-100 | 1.8×108CFU | 2+ | 0/4 | 4/4d |
| J45-Cr | 1.7×108CFU | 1+ | 0/2 | 2/2d |
| a从尸检肺样品回收的攻击菌株受J45-100攻击的猪在处理后4天解剖验尸。 | ||||
| b这一剂量是以前报导的50％致死剂量(Inzana，1993a)的6.6倍(5×106 CFU) | ||||
| c本组攻击后36小时内全部死亡 | ||||
| d从肺样品回收的胸膜肺炎芽孢杆菌无虹彩，并且不与第5血清型乳胶珠发生凝集反应，证明是无荚膜型的 | ||||
| e尸检表明一只猪的死亡是由于剂量错误。 | ||||
| fJ45-C是化学诱变的无荚膜型突变子，其特征已为人所知。 | ||||
低剂量(1.45×107CFU)J45-100攻击的五头猪不表现猪胸膜肺炎任何临床症状，不形成肺损伤。给药四天后尸检肺部取样培养后无胸膜肺炎放线芽孢杆菌生长。以较高剂量(2.95×107CFU)J45-100攻击的五头猪中的两头，临床正常，尸检无肺损伤。这组中的一头猪表现中度呼吸困难，尸检有肺充血和轻度的出血(肺损伤度＝1+)。本组另外两头猪高度呼吸困难，尸检有胸膜炎和实变(肺损伤度＝2+)。只有从这两头肺损伤最严重的猪能培养得到胸膜肺炎放线芽孢杆菌。从这些猪回收的细菌不使第5血清型a乳胶珠凝集剂发生凝集反应。因此回收得的细菌仍是无荚膜型的，表明J45-100未在体内恢复变为产荚膜型。
当将npt1(赋予细菌卡那霉素抗性)和sacB/sacR(赋予细菌蔗糖敏感性)基因被克隆至缺失位点时，这些基因仅作为标记基因。可选择使用其他标记基因。为健康和安全考虑，优选避免使用象npt1这样的抗生素抗性标记，或者应提供处理或使抗生素标记失活的机制。合适的非抗生素标记包括汞抗性标记。
按照以上步骤得到的第5血清型胸膜肺炎放线芽孢杆菌的无荚膜型菌株只产生胸膜肺炎放线芽孢杆菌所产三种毒素中的两种。该经修饰的胸膜肺炎放线芽孢杆菌是保护性的并能引起免疫反应，所以将第三个毒素基因RTX克隆到缺失位点可能有应用意义。可以通过将RTX毒素基因克隆到J45-100的卡那霉素基因盒，使卡那霉素基因失活来实现这一目的。
优选疫苗以与本领域已知疫苗相似的形式提供。优选疫苗是瓶装冻干物，可以包括一或多种血清型的突变菌株。为保持活性，应包括Columbia肉汤、海藻糖或清蛋白、甘油或其他试剂。冻干物成分只需用无菌水或盐水溶解，并注射(肌内、静脉、腹膜内、皮下等)。疫苗也可用合适的媒介(如淀粉、多糖、油、脂质体、树胶等)做成适应其他施用方式(如口服、经眼、舌下等)的形式。
疫苗给予动物的剂量由动物年龄、性别及给药方式等因素决定。总之，有效剂量的活的无毒无荚膜型胸膜肺炎放线芽孢杆菌应能在接种动物中引起免疫应答。用109菌落单位免疫两次，两次免疫间隔2到3周，可得到良好的效果。
本发明对优选实施方案进行了描述，本领域技术人员应认识到实施改进的发明仍在附具的权利要求的精神和范围内。
Claims (4)
1.一种疫苗，包含重组的、无毒、无荚膜胸膜肺炎放线芽孢杆菌，该菌缺少编码荚膜合成的DNA序列，其中所述编码荚膜合成的DNA序列是通过同源DNA序列间的重组所实现的等位基因交换而被缺失掉的。
2.一种重组的无荚膜胸膜肺炎放线芽孢杆菌的用途，所述杆菌用于制备对动物进行免疫以使之免患胸膜肺炎的疫苗，其中所述杆菌缺少编码荚膜合成的DNA序列，并且所述编码荚膜合成的DNA序列是通过同源DNA序列间的重组所实现的等位基因交换而被缺失掉的。
3.权利要求2的用途，其中所述疫苗为以肌肉内或皮下注射形式施用的疫苗。
4.一种制备疫苗的方法，所述疫苗用于防止由胸膜肺炎放线芽孢杆菌引起的疾病，该方法包括如下步骤：
识别出所述胸膜肺炎放线芽孢杆菌中编码荚膜合成的基因；和
通过同源DNA序列间的重组所实现的等位基因交换而使所述编码荚膜合成的基因缺失至少一部分，从而产生出无荚膜的胸膜肺炎放线芽孢杆菌菌株。
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| CA2530779C (en) * | 2003-07-02 | 2012-06-05 | Biotechnology Research And Development Corporation | Acapsular p. multocida hyae deletion mutants |
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| TWI548746B (zh) * | 2009-08-06 | 2016-09-11 | 英特威特國際股份有限公司 | 防備豬胸膜肺炎之疫苗及製備該疫苗之方法 |
| CN106866800B (zh) * | 2016-09-14 | 2020-08-18 | 四川农业大学 | 胸膜肺炎放线杆菌的体内诱导抗原及其应用 |
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| CZ295438B6 (cs) | 2005-08-17 |
| BR9710986A (pt) | 2001-12-11 |
| AR008615A1 (es) | 2000-02-09 |
| CA2258168A1 (en) | 1997-12-31 |
| NO986099D0 (no) | 1998-12-23 |
| EP0964689A1 (en) | 1999-12-22 |
| WO1997049416A1 (en) | 1997-12-31 |
| JP2001522347A (ja) | 2001-11-13 |
| SK177198A3 (en) | 2000-03-13 |
| CZ418998A3 (cs) | 1999-07-14 |
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| EP0964689A4 (en) | 2004-09-08 |
| NO986099L (no) | 1999-01-13 |
| US6326001B1 (en) | 2001-12-04 |
| PL186728B1 (pl) | 2004-02-27 |
| AU3390497A (en) | 1998-01-14 |
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