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1、SjFer与GM-CSF共表达真核表达载体诱导小鼠免疫保护效果农业生物技术JoumalofAgriculturalBiotechnology2007,15(4):567-573研究论文?SjFer与GM-CSF共表达真核表达载体诱导小鼠免疫保护效果陈尔曼,黄复深l,2料,邱元,郝知友,袁鑫(1.湖南农业大学动物医学院,长沙410128;2.湖南农业大学细胞工程重点实验室,长沙410128):l:摘要:根据GenBank中SjFer序列设计1对特异引物,以日本血吸虫(Schistosomajaponicum)cDNA文库为
2、模板扩增SjFer基因,常规方法构建质粒pcDNA3.O/SjFer,pcDNA3.O/GM-CSF及pcDNA3.O/SjFer-GM-CSFo85只昆明小鼠分A,B,C,D和E组,分别于0,2和4周通过左后肢股四头肌注射生理盐水50L/只,质粒50g/只.感染前和每次免疫前采血收集血清,ELISA检测特异性IgG水平.末次免疫后2周每组随机宰杀小鼠5只无菌取脾,MTT法检测淋巴细胞增殖情况,取免疫部位的肌肉组织通过免疫组化检查重组质粒在小鼠肌细胞内表达情况.末次免疫后2周每只小鼠贴壁感染40±1条尾蚴,45d后宰杀观
3、察减虫牢和减卯牢.结果表明,重组质粒能在小鼠肌细胞内表达;A,B和c组IgG水平变化不明显,D组和E组可诱导小鼠IgG水平升高,且E组高于D组.淋巴细胞转化试验结果A,B和c组OD5变化不明显,彼此尤显着差异,D组和E组脾淋巴细胞转化率明显增加,与对照组差异显着,且E组O明显高于D组.D组和E组可诱导小鼠产生36.27%和39.22%的减虫率,36.10%和49.04%的减卯率.GM-CSF可增强体液和细胞免疫应答并显着增强pcDNA3.O/SjFer的免疫保护力.关键词:日本血吸虫;铁蛋白基因;粒细胞集落刺激因子;DN
4、A疫苗;免疫保护力中图分类号:S188文献标识码:A文章编号:1006—1304(2007)一04—0567—7ImmunoprotectionElicitedbyEucaryoticExpressionVectorofCo—encodingSjFerandGM-CSFinMiceCHENEr—man,HUANGFu—sheng.料,QIUYuan.,HAOZhi—you,YUANXin.fJ.VeterinaryFaculty,HunanAgriculturalUniversity,Changsha410128,Chi
5、na;2.KeyLaboratoryofCellEngineering,HtmanAgriculturalUniversity,Changsha410128,Chh~a.)Abglr~Totestimmunoprotectionelicitedbyeucaryoticexpressionvectorofco—encodingSjFerritingene(SjFer)andgranulocytemacrophagecolony—stimulatingfactor(GM-CSF)againstchallengeinfecti
6、onbySchistosomajaponicuminmice,thespecialprimersweredesignedaccordingtothesequenceofSjFerinGenBank,andSjFerwasamplifiedfromS.japonicumadultwormcDNAlibrary.Ei曲ty—fivefemalemicewererandomlydividedinto5groups,immunizedwith50Lnormalsalt,50txgpcDNA3.0,5OgpcDNA3.O/GM-C
7、SF,50txgpcDNA3.O/SjFerand50gpcDNA3.O/SjFer-GM-CSFpermousein0,2ndand4thweek,respectively.Serawerecollectedbeforeimmunizationandchallengeinfection,respectively.LevelsofspecificantibodyweredetectedbyELISA.Lymph—spleencytesofmicewerecollectedtwoweeksafterfinalimmuniz
8、ation.ActivityofLymph—spleencyteproliferationwasdetectedbyMTTassay.Expressionofrecombinantplasmidsinmurinemusculartissuewasdetectedwithimmunohistochemistry.Mic
