【作者】 张志强；

【导师】 凌飞；

【作者基本信息】 西北农林科技大学 ， 水产养殖， 2025， 硕士

【摘要】 大口黑鲈作为我国重要的淡水养殖经济鱼类,其养殖规模持续扩大。然而,随着养殖密度的不断提升,大口黑鲈的病害问题也日益凸显,尤其是大口黑鲈虹彩病毒（LMBV）病,已成为对养殖业构成重大威胁的主要疾病之一。LMBV感染可引发大口黑鲈体表溃疡、内脏病变以及高死亡率,对养殖业造成严重的经济损失。目前,缺乏有效的治疗药物和疫苗,这已成为制约大口黑鲈产业可持续发展的关键因素。针对病毒蛋白靶标的抗病毒药物筛选是当前药物开发的重要方向,因此,建立一个靶向LMBV蛋白的体外药物筛选方法,以快速发现具有抗LMBV活性的药物,对于促进大口黑鲈产业的健康发展具有重要意义。解旋酶作为高度保守的病毒特异性蛋白,其功能位点（如ATP结合域、核酸解旋活性中心）是理想的药物靶标。本研究的目的是构建一个靶向LMBV解旋酶的体外药物筛选方法,并利用该方法筛选出具有抗LMBV活性的天然分子,为开发新型抗LMBV药物奠定基础。本研究获得的主要成果如下:1.LMBV解旋酶的原核表达系统及解旋反应体系的构建从LMBV全基因组序列中获取LMBV解旋酶基因序列,全长1215 bp,并将其克隆到原核表达载体p ET-28a中,构建p ET28a-Helicase表达载体。将表达载体转化到大肠杆菌Rosetta-gami 2（DE3）菌株中,优化表达条件,诱导时间设置为8 h,温度设置为16℃,IPTG浓度设置为l m M时,成功获得可溶性LMBV解旋酶重组蛋白。基于荧光共振能量转移方法,利用荧光标记的双链DNA底物,解旋后荧光信号发生变化建立解旋酶解旋反应体系。在设置底物F链为200 nM,Q链为240 nM的浓度下,LMBV解旋酶蛋白浓度设置为100 nM、解旋反应时间设置为40 min、解旋反应温度设置为32℃、ATP浓度设置为2 m M、C链浓度设置为800 nM、Mg²⁺设置为10 m M时,解旋酶活性最高,以此条件建立解旋酶解旋反应体系。利用该体系,通过检测荧光值的变化,可以评估化合物对LMBV解旋酶活性的抑制作用。2.靶向LMBV解旋酶的抗病毒分子筛选利用构建的LMBV解旋酶活性测定体系,对虚拟筛选和实验室保存的化合物进行筛选,除了阳性对照疱疹病毒解旋酶抑制剂普瑞利韦和阿莫那韦外,筛选出5种对LMBV解旋酶活性具有抑制作用的单体化合物,这7个化合物普瑞利韦、阿莫那韦、木犀草素、脱水穿心莲内酯琥珀酸半酯、鬼臼毒素、胆红素、橄榄苦甙对LMBV解旋酶的抑制率依次为87%、78%、82%、81%、80%、79%、77%。同时检测了这7个化合物普瑞利韦、阿莫那韦、木犀草素、脱水穿心莲内酯琥珀酸半酯、鬼臼毒素、胆红素、橄榄苦甙抑制LMBV解旋酶的IC₅₀依次为4.038μg/mL、5.01μg/mL、4.239μg/mL、4.766μg/mL、4.9μg/mL、4.658μg/mL、5.173μg/mL。其中IC₅₀最低的三个化合物依次为普瑞利韦、木犀草素、胆红素。3.抗LMBV分子活性验证评估筛选出的化合物对大口黑鲈脑上皮细胞（Bass Brain Cell,BBC）的细胞毒性,测得这7个化合物普瑞利韦、阿莫那韦、木犀草素、脱水穿心莲内酯琥珀酸半酯、鬼臼毒素、胆红素、橄榄苦甙的安全浓度依次为0.25μg/mL、0.5μg/mL、10μg/mL、10μg/mL、0.05μg/mL、10μg/mL、10μg/mL。并检测它们对LMBV感染的抑制效果,结果显示它们对LMBV MCP基因表达抑制率依次为86.8%、70.7%、96.0%、42.8%、80.0%、99.5%、24.0%。最终,胆红素表现出最佳的抗病毒活性,其IC₅₀值为4.348μg/mL,对LMBV MCP基因表达的抑制率最高可达99.6%。综合抑酶试验与抗病毒活性验证试验结果表明,胆红素是一种具有潜力的抗LMBV天然产物。综上所述,本研究成功构建了靶向LMBV解旋酶的体外药物筛选方法,并筛选出具有抗LMBV活性的单体化合物胆红素,为大口黑鲈虹彩病毒病的防控提供了新的思路和方法。更多还原

【Abstract】 Micropterus salmoides,as an important freshwater aquaculture economic fish in China,has seen a continuous expansion in its farming scale.However,with the increasing density of aquaculture,the disease problems of Micropterus salmoides have become increasingly prominent,especially the Micropterus salmoides iridovirus(LMBV)disease,which has become one of the major diseases posing a significant threat to the aquaculture industry.LMBV infection can cause skin ulcers,visceral lesions,and high mortality inMicropterus salmoides,causing severe economic losses to the aquaculture industry.Currently,there are no effective therapeutic drugs or vaccines,which has become a key factor restricting the sustainable development of the Micropterus salmoides industry.The screening of antiviral drugs targeting viral protein targets is an important direction in current drug development.Therefore,establishing an in vitro drug screening method targeting LMBV proteins to rapidly discover drugs with anti-LMBV activity is of great significance for promoting the healthy development of the Micropterus salmoides industry.Helicase,as a highly conserved virus-specific protein,has ideal drug targets in its functional sites(such as ATP binding domain,nucleic acid helicase active center).The main achievements of this study are as follows:1.Construction of the prokaryotic expression system and helicase reaction system of LMBV helicaseThe LMBV helicase gene sequence was obtained from the LMBV whole genome sequence,with a full length of 1215 bp,and was cloned into the prokaryotic expression vector p ET-28a to construct the p ET28a-Helicase expression vector.The expression vector was transformed into the Escherichia coli Rosetta-gami 2(DE3)strain,and the expression conditions were optimized.When the induction time was set at 8 h,the temperature at 16℃,and the IPTG concentration at 1 m M,the soluble LMBV helicase recombinant protein was successfully obtained.Based on the fluorescence resonance energy transfer method,a helicase helicase reaction system was established using fluorescently labeled double-stranded DNA substrates,where the fluorescence signal changes after helicase.When the substrate F chain concentration was set at 200 nM,the Q chain at 240 nM,the LMBV helicase protein concentration at 100 nM,the helicase reaction time at 40 min,the helicase reaction temperature at 32℃,the ATP concentration at 2 m M,the C chain concentration at 800 nM,and the Mg2+at 10 m M,the helicase activity was the highest.Under these conditions,the helicase helicase reaction system was established.Using this system,the inhibitory effect of compounds on LMBV helicase activity can be evaluated by detecting the change in fluorescence value.2.Antiviral molecular screening targeting LMBV helicaseUsing the constructed LMBV helicase activity assay system,virtual screening and laboratory-stored compounds were screened.In addition to the positive controls,the herpesvirus helicase inhibitors pritelivir and amonafide,five monomer compounds with inhibitory effects on LMBV helicase activity were screened out.The inhibition rates of these seven compounds,pritelivir,amonafide,luteolin,dehydro-andrographolide succinate hemiesters,podophyllotoxin,bilirubin,and oleuropein,on LMBV helicase were 87%,78%,82%,81%,80%,79%,and 77%,respectively.The IC50 values of the seven compounds,pralivir,amonavir,luteolin,andrographolide succinate hemiesters,podophyllotoxin,bilirubin and oleuropein in inhibiting LMBV helicase were 4.038μg/mL,5.01μg/mL,4.239μg/mL,4.766μg/mL,4.9μg/mL,4.658μg/mL and 5.173μg/mL respectively.Among them,the three compounds with the lowest IC50 values were pralivir,luteolin and bilirubin in sequence.3.Verification of anti-LMBV drug activityThe cytotoxicity of the selected compounds to bass brain cells(BBC)was evaluated,and the safe concentrations of the seven compounds pralivir,amonavir,luteolin,andrographolide succinate hemiesters,podophyllotoxin,bilirubin and oleuropein were 0.25μg/mL,0.5μg/mL,10μg/mL,10μg/mL,0.05μg/mL,10μg/mL and 10μg/mL respectively.Their inhibitory effects on LMBV infection were also detected.The results showed that the inhibition rates of LMBV MCP gene expression were 86.8%,70.7%,96.0%,42.8%,80.0%,99.5%and 24.0%respectively.Finally,bilirubin showed the best antiviral activity,with an IC50 value of 4.348μg/mL and the highest inhibition rate of LMBV MCP gene expression up to 99.6%.The results of the enzyme inhibition test and the antiviral activity verification test indicated that bilirubin is a potential natural product against LMBV.In conclusion,this study successfully established an in vitro drug screening method targeting LMBV helicase and screened out the monomer compound bilirubin with anti-LMBV activity,providing new ideas and methods for the prevention and control of LMBV disease in largemouth bass.更多还原