红螯螯虾卵黄蛋白原VWD结构域的原核表达及纯化

【目的】原核表达红螯螯虾（Cherax quadricarinatus）卵黄蛋白原（Vg）VWD结构域，为深入开展Vg的生物学功能研究和开发相应的生物活性物质提供技术支持，也为改进虾类养殖催熟及获取高质量后代打下基础。【方法】在GenBank中搜索红螯螯虾卵Vg的mRNA序列（AF306784.1）及查找其VWD结构域（2345~2491 aa），采用ExPASyProtParam、ProtScale、InterProscan及TMHMM等在线软件进行生物信息学分析及理论评估，结合大肠杆菌密码子偏好优化原始VWD氨基酸序列；然后通过全基因合成获得目的基因，连接至载体pET-28a构建重组质粒pET-28a-VWD，挑取阳性克隆转化大肠杆菌BL21（λDE3）感受态细胞及采用IPTG进行诱导表达，并以10% SDS-PAGE电泳和Western blotting检测诱导表达的融合蛋白。【结果】红螯螯虾VWD结构域相对分子量为19692.11 Da，理论等电点（pI）为9.35，分子式为C₈₅₅H₁₃₃₄N₂₅₈O₂₆₁S₉，属于不稳定的亲水性蛋白，不含跨膜结构域。延伸链和无规则卷曲是红螯螯虾VWD结构域二级结构的主要元件，其中，α-螺旋占7.73%，β-转角占10.50%，延伸链占35.91%，无规则卷曲占45.86%。重组质粒pET-28a-VWD经大肠杆菌BL21（λDE3）感受态细胞诱导表达即获得融合蛋白VWD，以2 mol/L盐酸胍溶解和Ni-NTA亲和层析纯化及复性后，10% SDS-PAGE电泳和Western blotting检测均在蛋白相对分子量约20.0 kD处出现1条清晰的特异性条带，融合蛋白VWD表达形式以包涵体为主。融合蛋白VWD在BL21（λDE3）感受态细胞中高效表达的最佳诱导条件:当单克隆菌液OD_{600 nm}达0.6时添加0.5 mmol/L IPTG，置于20℃下诱导培养16 h。纯化后的融合蛋白VWD浓度为1.32 mg/mL。【结论】通过全基因合成获得的优化红螯螯虾VWD基因能在大肠杆菌BL21（λDE3）感受态细胞中诱导表达出以包涵体为主要形式的融合蛋白，经盐酸胍溶解和Ni-NTA亲和层析柱纯化及复性即可获得高纯度的活性VWD蛋白，为后续开展红螯螯虾Vg生物学功能研究及开发相应的生物活性物质提供技术支持。

Prokaryotic expression and purification of VWD domain in vitellogenin of Cherax quadricarinatus

【Objective】Prokaryotic expression of the vitellogenin(Vg) VWD domain of red claw crayfish(Cherax quadricarinatus) not only provided technical support for further research on the biological function of Vg and the development of corresponding bioactive substances, but also laid foundation for improving C. quadricarinatus culture, accelerating ripening and obtaining high-quality offspring.【Method】The protein sequence of VWD domain(2345-2491 aa) was found according to the Vg mRNA sequence (accession number:AF306784.1) of C. quadricarinatus published in GenBank database. Bioinformatics analysis and theoretical evaluation were carried out by using online softwares such as ExPASyProtParam, ProtScale, InterProscan and TMHMM. Optimization of original VWD amino acid sequence combined with codon preference of Escherichia coli. Then the target gene was obtained by whole gene synthesis, and the recombinant plasmid E. coli pET-28a-VWD was constructed by connecting to vector pET-28a, and transformed into positive clone conversion BL21 (λDE3) competent cells which were induced by IPTG. The induced fusion protein was detected by 10% SDSPAGE electrophoresis and Western blotting.【Result】The relative molecular weight of VWD domain of C. quadricarinatus was 19692.11 Da, theoretical isoelectric points(pI) was 9.35, and the molecular formula was C₈₅₅H₁₃₃₄N₂₅₈O₂₆₁S₉. It was an unstable hydrophilic protein and did not contain a transmembrane domain. Extended chain and irregular curl were the main elements of the secondary structure of VWD domain in C. quadricarinatus, among which, α-helix accounted for 7.73%, β-turn accounted for 10.50%, the extended chain accounted for 35.91%, and the irregular curl accounted for 45.86%. The recombinant plasmid pET-28a-VWD expressed the fusion protein VWD by inducing the competent cells E. coli BL21(λDE3). After dissolution with 2 mol/L guanidine hydrochloride and purification and renaturation on Ni-NTA affinity chromatography column, a clear specific band appeared at the relative molecular weight of the protein about 20.0 kD by 10% SDS-PAGE electrophoresis and Western blotting. The expression form of fusion protein VWD was mainly inclusion body. The best induction conditions for high expression of the fusion protein VWD in the competent cells BL21 (λDE3):when the OD_{600 nm} of the monoclonal bacterial solution reached 0.6, 0.5 mmol/L IPTG was added and cultured at 20℃ for 16 h. The concentration of purified fusion protein VWD was 1.32 mg/mL.【Conclusion】The optimized VWD gene of C. quadricarinatus obtained by whole gene synthesis can be expressed in E. coli competent cells BL21(λDE3), and the expression form of fusion protein VWD is mainly inclusion body. After dissolving with guanidine hydrochloride and purifying and renaturating with Ni-NTA affinity chromatography column, high-purity active VWD protein can be obtained, which provides technical support for the subsequent research on the biological function of C. quadricarinatus Vg and the development of corresponding bioactive substances.