克氏原螯虾新型凝集素PcLT的重组表达及应用

doi:10.16431/j.cnki.1671-7236.2025.08.034

摘要/Abstract

摘要： 【目的】对克氏原螯虾新型凝集素PcLT基因序列进行密码子优化，利用生物信息学软件分析该序列编码蛋白的理化性质及结构，通过原核表达系统表达PcLT重组蛋白，并评估其在虾白斑综合征病毒病中的应用效果。【方法】从GenBank中获取PcLT基因序列(登录号:JQ670880.1)，以大肠杆菌密码子偏好对其进行优化，并利用生物信息学软件预测蛋白理化性质及二级结构、三级结构等。通过T4连接酶将PcLT基因片段与pET-32a(＋)载体连接构建重组质粒pET32a-PcLT，并利用大肠杆菌BL21(DE3)原核表达系统表达重组蛋白PcLT，使用Ni柱亲和层析法进行蛋白纯化，将重组蛋白PcLT与虾料混合饲喂感染白斑综合征病毒(White spot syndrome virus，WSSV)的病虾，检测其对虾白斑综合征病毒病的应用效果。【结果】 PcLT基因编码169个氨基酸，PcLT蛋白为稳定、疏水性蛋白，理论等电点为4.17，溶解度为0.645；该蛋白为非跨膜蛋白，含有27个磷酸化位点，有分泌性信号肽；PcLT蛋白二级结构主要包括α-螺旋(26.63%)、延伸链(21.30%)和无规则卷曲(52.07%)，三级结构主要以无规则卷曲为主。成功构建pET32a-PcLT重组质粒，表达并纯化了重组蛋白PcLT，其分子质量为33 ku；100 mL PcLT重组蛋白饲喂病虾5 d后，虾体内的白斑综合征病毒拷贝数下降，病毒检测结果由阳性转为阴性，试验虾吃料正常。【结论】试验成功表达重组蛋白PcLT，其在抗虾白斑综合征病毒应用中效果良好，研究结果为虾养殖中的白斑综合征病毒防治及相关研究提供理论基础。

关键词: 克氏原螯虾; 新型凝集素PcLT; 白斑综合征病毒

Abstract: 【Objective】 The codon of the novel lectin PcLT gene sequence of Procambarus clarkii was optimized，and the physicochemical properties and structure of the protein encoded by this sequence were analyzed using bioinformatics software.The recombinant PcLT protein was expressed through the prokaryotic expression system,and the application effect in white spot syndrome virus disease of shrimp was evaluated. 【Method】 The PcLT gene sequence was obtained from GenBank (accession No.：JQ670880.1),optimized according to the codon preference of Escherichia coli.The physicochemical properties,secondary structure and tertiary structure of the protein were predicted by bioinformatics software.The PcLT gene fragment was ligated with the pET-32a(＋) vector by T4 ligase to construct the recombinant plasmid pET32a-PcLT.The recombinant protein PcLT was expressed in prokaryotic expression system of Escherichia coli BL21 (DE3).The protein was purified by Ni-column affinity chromatography.The recombinant protein PcLT was mixed with feed and fed to diseased shrimp infected with White spot syndrome virus (WSSV)，and to detect its application effect. 【Result】 PcLT gene encoded 169 amino acids，and PcLT protein was a stable and hydrophobic protein with a theoretical isoelectric point of 4.17 and a solubility of 0.645.It was a non-transmembrane protein,containing 27 phosphorylation sites and a secretory signal peptide.The secondary structure of PcLT protein mainly included alpha helix (26.63%),extended chain (21.30%) and random coil (52.07%),and the tertiary structure was mainly random coil.The recombinant plasmid pET32a-PcLT was successfully constructed,and the recombinant protein PcLT was expressed and purified.The molecular mass of PCLT protein was 33 ku.After feeding the shrimp with 100 mL PcLT recombinant protein for 5 days,the copy number of WSSV in the shrimp decreased,the virus detection result turned from positive to negative,and the shrimp ate the feed normally. 【Conclusion】 The recombinant protein PcLT was successfully expressed,and it had a good effect in the application against WSSV.This results provided a theoretical basis for the prevention and treatment of WSSV in shrimp aquaculture and related research.

Key words: Procambarus clarkii; novel lectin PcLT; White spot syndrome virus

中图分类号:

S851.34^＋7.34

于洪敏, 李娟, 王岩, 徐艺娜, 刘艳. 克氏原螯虾新型凝集素PcLT的重组表达及应用[J]. 中国畜牧兽医, 2025, 52(8): 3857-3864.

YU Hongmin, LI Juan, WANG Yan, XU Yina, LIU Yan. Recombinant Expression and Application of Novel Lectin PcLT in Procambarus clarkii[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3857-3864.

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