CAJ | 학술논문

目的：选用人巨细胞病毒( HCMV)抗原性强且亲水性较高片段构建融合抗原表达质粒，诱导其表达，纯化目的蛋白；并初步鉴定其免疫反应性。方法通过Protean软件分析了HCMV UL32、UL44、UL83序列，设计合理引物；各基因片段用Overlap方法连接，并引入BglⅡ、BamHⅠ酶切位点，使之可通过酶切与质粒pQE-80L连接。重组质粒转化入感受态BL21(DE3)菌株，经PCR、酶切鉴定目的基因后再诱导、表达、纯化，获得目的蛋白，采用Western blot法检测免疫反应性。结果诱导后的重组菌pQE80-L-UL32-UL44-UL83经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳( SDS-PAGE )证实蛋白表达在包涵体。纯化后获得大小约为52 ku的目的蛋白，采用Western blot法检测证实重组嵌合抗原能与HC-MV阳性血清有反应。结论构建了重组嵌合抗原表达质粒，并诱导、表达、纯化获得目的蛋白。 Western blot法鉴定嵌合抗原是HCMV特异性抗原且有免疫反应性。
목적：선용인거세포병독( HCMV)항원성강차친수성교고편단구건융합항원표체질립，유도기표체，순화목적단백；병초보감정기면역반응성。방법통과Protean연건분석료HCMV UL32、UL44、UL83서렬，설계합리인물；각기인편단용Overlap방법련접，병인입BglⅡ、BamHⅠ매절위점，사지가통과매절여질립pQE-80L련접。중조질립전화입감수태BL21(DE3)균주，경PCR、매절감정목적기인후재유도、표체、순화，획득목적단백，채용Western blot법검측면역반응성。결과유도후적중조균pQE80-L-UL32-UL44-UL83경십이완기류산납-취병희선알응효전영( SDS-PAGE )증실단백표체재포함체。순화후획득대소약위52 ku적목적단백，채용Western blot법검측증실중조감합항원능여HC-MV양성혈청유반응。결론구건료중조감합항원표체질립，병유도、표체、순화획득목적단백。 Western blot법감정감합항원시HCMV특이성항원차유면역반응성。
Objective To construct fusion antigen expression plasmid by choosing strong antigenicity and higher hydrophilic segments of HCMV gene, we induce expression, purify target protein and preliminarily identify immu-nogenicity of HCMV in order to develop fast and sensitive diagnostic kits research laboratory diagnosis. Methods in future. Methods Sequences of HCMV UL-32,UL-44,UL-83, were analyzed by software of Protean to design rea-sonable primers. Each gene fragment was linked by using Overlap method at the same time PstⅠ, BglⅡ,BamHⅠsites were introduced to make it connected to plasmid pQE80-L by enzyme digestion, then the recombinant plasmid was transformed into BL21. Target gene identified by PCR and digestion was induced to express the protein. The protein was purified and its immunoreactivity was assayed by Western blot. Results After inducing, recombinant bacteria, pQE80-L-UL32-UL44-UL83, had a fusion of 52 ku protein by SDS-PAGE electrophoresis. Recombinant chimeric antigen could react to HCMV positive serum through Western blot. Conclusion The recombinant fusion antigen expression plasmid is constructed to obtain target protein. Multi-epitope antigen is confirmed to be the spe-cific antigen for HCHV and has immunity by Western blot.