目的：利用昆虫杆状病毒表达系统（昆虫BEVS）表达糖酵解酶α-烯醇化酶（ENO1）及其3种酶活性位点缺失突变的 ENO1 蛋白 ENO1-M1、ENO1-M2 和 ENO1-M3，为后续宫颈癌的代谢治疗研究奠定基础。方法：利用分子克隆技术将优化后 ENO1序列插入pFastBacTM1载体，获得含有目的基因的重组质粒 pFastBac-ENO1。分别缺失ENO1发挥糖酵解酶功能的3个活 性位点，进行优化后将其插入pFastBacTM1载体，获得3个活性位点缺失的重组质粒pFastBac-M1、pFastBac-M2和pFastBac-M3。 通过转座、转染后获得重组杆状病毒rBV-ENO1、rBV-M1、rBV-M2和rBV-M3，利用WB法对目的蛋白的表达及特异性进行检测。结 果：成功扩增重组杆粒rBacmid-ENO1、rBacmid-M1、rBacmid-M2和rBacmid-M3，获得大小约2 000 bp的基因片段，与预期大小相 符。昆虫BEVS可表达ENO1蛋白及其3个酶活位点缺失的重组蛋白ENO1-M1、ENO1-M2和ENO1-M3，其分子量约为52 000，与 预期相符。WB法鉴定这些蛋白能与特异性标签His-tag发生反应。结论：通过昆虫BEVS成功表达目的蛋白ENO1及其酶活性 位点缺失蛋白ENO1-M1、ENO1-M2和ENO1-M3，这些蛋白具有反应原性，为后续测定这些蛋白与ENO1单抗亲和力创造了条件。

Objective: To express the glycolytic enzyme alpha-enolase (ENO1) and its three enzyme active site deletion mutant ENO1 proteins (ENO-M1, pFastBac-M2, ENO1-M3) using a baculovirus expression vector system (insect BEVS), laying the groundwork for the subsequent study of metabolic therapy for cervical cancers. Methods: Molecule cloning was used to insert optimized ENO1 gene sequence into the pFastBacTM1 vector to obtain the recombinant plasmid pFastBac-ENO1 with target genes. Three active sites essential for ENO1's glycolytic function were deleted, and the corresponding optimized sequences were inserted into the pFastBacTM1 vector to generate the recombinant plasmids with three active site deletion, namely pFastBac-M1, pFastBac-M2 and pFastBac-M3. Recombinant baculoviruses rBV-ENO1, rBV-M1, rBV-M2 and rBV-M3 were subsequently obtained through transposition and transfection. The expression and specificity of the target proteins were examined using WB assay. Results: Recombinant bacilli rBacmid-ENO1, rBacmid-M1, rBacmid-M2 and rBacmid-M3 were successfully amplified, obtaining a gene fragment of about 2 000 bp in size, which was consistent with the expected size. The insect BEVS could express the ENO1 protein and its recombinant proteins (ENO1-M1, ENO1-M2, ENO1-M3) with three enzyme active site deletions, each with a molecular weight of approximately 52 000, as expected. WB analysis confirmed that these proteins reacted with the specific His-tag antibody. Conclusion: The insect BEV successfully expresses the target protein and its proteins with enzyme active site deletions, namely ENO1-M1, ENO1-M2 and ENO1-M3. This protein's reactivity establishes the foundation for subsequent determination of the affinity of these proteins, and ENO1 monoclonal antibody.

国家自然科学基金（No. 82260557）