疫苗级乳球菌表达载体的构建及柔嫩艾美耳球虫TA4基因的表达

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1、中国农业大学博士学位论文疫苗级乳球菌表达载体的构建及柔嫩艾美耳球虫TA4基因的表达姓名：张莉申请学位级别：博士专业：预防兽医学指导教师：刘尚高;秦泽荣2002.5.1据已发表的球虫TA4基因序列设计特异性引物，并在引物的两端分别加上KpnI和SacI作为插入的酶切位点，将TA4基因克隆到表达载体pMG36n中，电穿7L法转化乳酸乳球菌，获得有TA4抗原活性的乳酸乳球菌工程菌，对该菌的表达产物用SDS—PAGE检测，结果表明表达产物的蛋白分子量为25KDa，与预期结果相同。以刚出壳的健康雏鸡为试验对象，用上述表达球虫TA4基因的工程乳球菌口服雏鸡10天，之后进行人工感染

2、球虫试验，一周后宰杀，通过试验鸡体重、粪便中卵囊数、盲肠病变记分、盲肠卵囊数和抗球虫指数等指标进行评价，结果抗球虫指数为160．12，达到良好水平。一连●州扩，忖0．，镰因一k{l』，AT篮臻体载达潦。1：：I、f，，菌““球；乳∽挣刀够=}丑疫词键关AbstractNumerousvectorshavebeendevelopedforgenetictransfer．Traditionally,themajoritycontainsselectablemarkersencoingresistancetoantibioticsusedinhumandrugtherapy

3、,butthiswouldbeundesirableinfood-producingmicroorganismsbecauseofthepossibletransferofantibioticresistancetothehumanintestinalmicroflora．Therefore，otherselectablemarkers，whichwouldbeconsideredsafeforuseinfood-gradecloningvectors，shouldbedevelopedandintroducedintofood—producingmicroorgan

4、isms．Ideally,afood—gradecloningvectorshouldbeconstructedfromDNAderivedfrommicroorganismsappmvedforfooduse，inducedbyfood-gradeinducers，andcontainsaselectablemarkerthatdoesnotcompromisehumandrug，herapy．However,thecomplementselectionmarkersalenotalwaysdesirable．Forexample，lactoserequiresth

5、edevelopmentofspecificstrainsthataredeficientinlactoseutilization．Sothedominantgeneticdeterminantsencodingpropertiessuch硒nisinresistanceshouldbeisolatedandclonedintovectorssuitableforgenetictransfer．Nisinisaribosomallysynthesizedant／bacterialpeptideproducedbycertainstrainsofLactococcusl

6、actis。whichiswidelyutilizedinfoOdindustry船highlyeffective，safeandnatul试foodpreservativebutnotusedinhumantherapy．Therefore，thenisinresistancemisr)phenotypeshouldbesafeforuse嬲aselectablemarkerinafood-gradecloningvector．Nisinresistancegenecallbeused硒aselectivemarkertoconstructlactococcalfo

7、od-gradevectors．Thispaper&scribesattemptstointroduceaNis‘markerintotheconstructionofcloningvectorSO鹊toaSsessitspotentialasafood-gradevectorforsubeloningtheresultinggeneta4．Accordingtothegeneticlociforcloselylinkednisin-resistance(Nis。)andlactose-fermentingability(Lao)，nisin-res