[目的]构建毛囊特异性表达绵羊毛角蛋白Ⅱ型中间丝9(KIF Ⅱ-9)cDNA的毛囊特异性表达载体,并证明其表达的有效性.[方法]根据绵羊毛角蛋白关联蛋白6.1(KAP6.1)基因已知DNA序列设计合成2个特异性引物,以绵羊基因组DNA为模板,通过PcR扩增得到KAP6-15'调控序列,然后与毛角蛋白Ⅱ型中间丝基因(KIF Ⅱ-9)CDNA、去除CMY启动子的表达载体pcDNA3.1连接构成皮肤特异性表达载体pcDNA3.1-KK,新生小鼠皮下注射该重组载体证明其表达有效性.[结果]PCR扩增出1 042 bp的特异性片段,DNA测序结果证明,该克隆片段与KAP6-1 5'端调控区序列相比具有极高的一致性;构建的毛囊特异性表达载体PCDNA3.1-KK,皮下注射新生小鼠,皮肤内KIF Ⅱ-9得到转录.[结论]该试验构建的载体PCDNA3.1-KK具有表达特异性.%[Objective] The aim was to construct recombinant follicle-specificexpression vector which can specifically express keratin type Ⅱ intermediate filament gene(KIF Ⅱ-9)in the hair follicle and prove its specificity.[Method] Two specific oligonucleotide primers were designed and synthesized according to the sequence of ovine keratin associated protein 6-1 (KAP6-1)gene, which were used to amplify the KAP6-1 5' regulating sequence,pcDNA3.1-KK,the follicle-specific expression vector, was constructed by connecting with KAP6-1 5' regulating sequence,KIF Ⅱ-9 cDNA and CMV promoter-less pcDNA3.1, and its expression specificity was proved by directly injecting the vector into the skin of newborn mice.[Result] A size of 1042 base pairs fragment was amplified and the sequencing results showed that the fragment has a high identity with original sequence.The recombinant expression vector was identified by PCR.The transient expression results indicated that the follicle-specific expression vector could efficiently direct the transcription of KIF Ⅱ-9 in skin.[Conclusion] The constructed vector pcDNA 3.1-KK has specific expression.
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