pcDNA3-HBsAg-p30-ROP2真核表达载体的构建与鉴定-论文.pdf

《pcDNA3-HBsAg-p30-ROP2真核表达载体的构建与鉴定-论文.pdf》由会员上传分享，免费在线阅读，更多相关内容在应用文档-天天文库。

1、·46·中国血吸虫病防治杂志2014年第26卷第1期ChinJSchistoControl2014，Vo1．26，No．1[文章编号]1005．6661(2014)01．0046-05·论著·pcDNA3．HBsAgp30一ROP2真核表达载体的构建与鉴定魏庆宽，王英婷。，闫运琴，肖婷，李瑾，徐超，刘功振，刘娟关，仲维霞，尹昆，付斌，闫歌，黄炳成[摘要]目的构建peDNA3一HBsAg—p30一ROP2多基因重组表达载体，并对其进行初步鉴定。方法根据重组体pcDNA3一p30一ROP2酶切位点和乙型肝炎表面抗原(HBsAg)基因序列等因素设计合成引物，扩

2、增HBsAg目的基因片段，再应用酶切、连接等分子生物学技术将HBsAg目的基凶克隆至peDNA3一p30一ROP2表达载体中。应用聚合酶链反应(PCR)初筛，冉采用酶切、测序等技术对构建的重组表达载体peDNA3一HBsAg—p30一ROP2进行鉴定。结果PCR扩增出HBsAg基【大J片段，构建了pcI)NA3一HBsAg—p30一ROP2多基因真核表达载体。PCR与酶切结果显示，该基因片段大小均与理论值相符；测序结果示该重组表达载体包含了p30一ROP2和HBsAg目的基因的完整序列。结论成功构建了多基因重组表达载体pcDNA3一HBsAg—p30一R

3、OP2，为进一步研究多基因核酸疫苗奠定了基础。[关键词]弓形虫；表面抗原1(p30)；棒状体分泌抗原2(ROP2)；乙肝表面抗原；基因重组[中图分类号]R382．5[文献标识码】AConstructionandidentificationofpcDNA3一HBsAg—p30一RoP2expressionvec·torWEIQing—kuan。。27WANGYing—ting，YANYun—qin，XIA0Ting，LIn，XUChao，LIUGong—zhen，ZHONGWei—xia．YINKun，FUBin‘，YANGe’，HUANGBing—che

4、ng1ShandongAcademyofMedicalSciences，ShandongInstituteofParasiticDiseases，ShandongAcademyofMedicalScienceKeyLaboratoryofMolecularImmunologyofParasiticDiseases，ShandongProvince，fining272033，China；2SchoolofMedicineand，JireSciences，UniversityofJinan—SandongAcademyofMedicalSciences，Sh

5、andongProvince，China；3FirstPeople’Hospitaloffining，ShandongProvince，China；4ShandongDaizhuangHospital，ShandongProvince，ChinaCorrespondingauthor【AbstractJObjectiveToconstructamulti—generecombinantpcDNA3一HBsAg—p30一ROP2expressionvectorandidenti~itpreliminarily．MethodsAccordingtorecom

6、binantpcDNA3一p30一ROP2restrictionsites，HBVHBsAggenesequencesofprimersweredesignedandsynthesizedtoamplifytargetfragment，andthenclonedintopcDNA3一HbsAg—p30一ROP2expressionvector．Af-tersequencing，itwasidentifiedfinallybyrestrictionenzymedigestionandothermolecularbiologytechniques．Resul

7、tsHBVHBsAggenesegmentwasamplifiedbyPCRandthemulti-generecombinantpcDNA3-HBsAg．-p30-ROP2expressionvectorwasconstructedandidentifiedtobecorrectastheoreticalvalues．ThePCRandrestrictionenzymedigestionresultsshowedthatHBsAgandp30一ROP2geneinrecombinantplasmidwereconfirmedbyDNAsequencin

8、g．ConclusionThemuhi—generecombinantpcD—N