白纹伊蚊唾液腺serpin1基因的可变剪接分析与原核表达

Alternative splicing and expression of the Alboserpin-1 from salivary gland of Aedes albopictus

目的克隆并原核表达白纹伊蚊唾液腺丝氨酸蛋白酶抑制剂基因1(Aalbserpin-1)。方法根据NCBI网站上白纹伊蚊Aalbserpin-1(AY826096.1)的序列设计引物,用RT-PCR从白纹伊蚊广州株中获取Aalbserpin-1全长基因序列,并进行生物信息学分析。将目的片段克隆到原核表达载体pET28a(+),转化到大肠杆菌中诱导表达。利用实时荧光定量RTPCR分析Aalbserpin-1_2基因在白纹伊蚊不同组织中的表达差异。结果 PCR扩增获得Aalbserpin-1基因两个转录本(Aalbserpin1_1和Aalbserpin1_2),Aalbserpin1_1的基因序列为1 260bp,编码419个氨基酸;Aalbserpin1_2的基因序列为1 332bp,编码443个氨基酸,均具有seprin结构域,Aalbserpin1_1和Aalbserpin1_2与Aalbserpin-1(AY826096.1)相比,相似性分别为95%和90%。两个转录本比较,Aalbserpin1_2中含有24个氨基酸的可变剪接子正好位于其功能活性中心环(RCL)上。以Aalbserpin1_2序列为模板,成功构建pET28a-Aalbserpin-1_2重组质粒。SDS-PAGE结果显示目的基因在Origami(DE3)中表达,重组蛋白相对分子量(Mr)约为50 000Da,经亲和层析获得目的蛋白。组织表达谱显示Aalbserpin-1_2在唾液腺(SG)、中肠(MG,P>0.05)丰富表达,脂肪体低表达(FB,P<0.05)。结论白纹伊蚊广州株Aalbserpin-1基因具有两个可变剪接子,其中Aalbserpin-1_2在唾液腺、中肠丰富表达,成功构建pET28a-Aalbserpin-1_2重组质粒并获得重组蛋白。

To clone and prokaryotically express the Aalbserpin-1 gene from the salivary gland of Aedes albopictus,the full-length cDNA sequence of Aalbserpin-1 gene was amplified by RT-PCR,then constructed into the prokaryotic expression vector pET28a（＋） and expressed in E.coli BL21（DE3） with IPTG induction.The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography.An expression analysis was conducted by real-time RT-PCR.It was demonstrated that two alternative transcripts were obtained （Aalbserpin-1_1 and Aalbserpin-1_2）.To compare with Aalbserpin-1 （AY826096.1）,Aalbserpin1_1 and Aalbserpin-1_2 shared 95% and 90% similarity respectively.An alternative splicing exon （24 amino acids：NLLTNRIVSQSSYRRVAIYDFISG） in Aalbserpin1_2 was just located in the reactive centre loop （RCL） of SERPIN,a primary determinant of functionality.A recombinant Aalbserpin-1_2 protein was purified and obtained.In addition,we also found Aalbserpin-1_2 was expressed higher in salivary gland and midgut than in fat body （P〈0.05）.The results suggest that the Aalbserpin-1 gene from Ae.albopictus exists two alternative transcripts,and Aalbserpin-1_2 is expressed highly in salivary gland and midgut,indicating that the rAalbserpin-1_2 protein has been prokaryotic expressed and purified successfully.