中国水产科学
中國水產科學
중국수산과학
Journal of Fishery Sciences of China
2014年
1期
10-18
,共9页
WSSV%囊膜蛋白VP110%结合蛋白%精氨酸激酶%中国明对虾%克氏原螯虾
WSSV%囊膜蛋白VP110%結閤蛋白%精氨痠激酶%中國明對蝦%剋氏原螯蝦
WSSV%낭막단백VP110%결합단백%정안산격매%중국명대하%극씨원오하
WSSV%envelope protein VP110%binding protein%arginine kinase%Fenneropenaeus chinensis%Pro-cambarus clarkia
为了鉴定对虾白斑病综合征病毒(WSSV)囊膜蛋白VP110在中国明对虾(Fenneropenaeus chinensis)鳃细胞中的结合蛋白,运用pET-32(a)+载体构建了1段含RGD模体的截短VP110原核重组表达质粒,转化大肠杆菌诱导表达后获得分子量为41 kD的截短重组VP110蛋白(rVP110)。以rVP110作为诱饵蛋白,运用pull-down实验结合蛋白质谱分析鉴定 rVP110结合蛋白,结果显示,中国明对虾鳃细胞中的肌动蛋白和精氨酸激酶(arginine kinase,AK)与rVP110具有结合作用。利用PCR扩增中国明对虾AK编码基因,将其与表达载体pGEX-4T-1连接后转化大肠杆菌诱导表达获得重组 AK 蛋白(rAK),通过 pull-down 实验进一步证实 rAK 可与 rVP110发生结合。克氏原螯虾(Procambarus clarkia)体内中和实验结果显示, rAK对WSSV感染克氏原螯虾具有一定的中和作用,能延缓螯虾的死亡进程。另外,中国明对虾在人工感染WSSV后,荧光定量PCR检测结果显示, AK基因表达水平显著上调,18 h时达到峰值,然后下降至正常水平；酶底物法检测结果同样显示,鳃细胞中AK酶活性在感染WSSV后发生显著上调。本研究旨在为深入了解WSSV囊膜蛋白VP110在WSSV感染宿主过程中的作用提供基础依据。
為瞭鑒定對蝦白斑病綜閤徵病毒(WSSV)囊膜蛋白VP110在中國明對蝦(Fenneropenaeus chinensis)鰓細胞中的結閤蛋白,運用pET-32(a)+載體構建瞭1段含RGD模體的截短VP110原覈重組錶達質粒,轉化大腸桿菌誘導錶達後穫得分子量為41 kD的截短重組VP110蛋白(rVP110)。以rVP110作為誘餌蛋白,運用pull-down實驗結閤蛋白質譜分析鑒定 rVP110結閤蛋白,結果顯示,中國明對蝦鰓細胞中的肌動蛋白和精氨痠激酶(arginine kinase,AK)與rVP110具有結閤作用。利用PCR擴增中國明對蝦AK編碼基因,將其與錶達載體pGEX-4T-1連接後轉化大腸桿菌誘導錶達穫得重組 AK 蛋白(rAK),通過 pull-down 實驗進一步證實 rAK 可與 rVP110髮生結閤。剋氏原螯蝦(Procambarus clarkia)體內中和實驗結果顯示, rAK對WSSV感染剋氏原螯蝦具有一定的中和作用,能延緩螯蝦的死亡進程。另外,中國明對蝦在人工感染WSSV後,熒光定量PCR檢測結果顯示, AK基因錶達水平顯著上調,18 h時達到峰值,然後下降至正常水平；酶底物法檢測結果同樣顯示,鰓細胞中AK酶活性在感染WSSV後髮生顯著上調。本研究旨在為深入瞭解WSSV囊膜蛋白VP110在WSSV感染宿主過程中的作用提供基礎依據。
위료감정대하백반병종합정병독(WSSV)낭막단백VP110재중국명대하(Fenneropenaeus chinensis)새세포중적결합단백,운용pET-32(a)+재체구건료1단함RGD모체적절단VP110원핵중조표체질립,전화대장간균유도표체후획득분자량위41 kD적절단중조VP110단백(rVP110)。이rVP110작위유이단백,운용pull-down실험결합단백질보분석감정 rVP110결합단백,결과현시,중국명대하새세포중적기동단백화정안산격매(arginine kinase,AK)여rVP110구유결합작용。이용PCR확증중국명대하AK편마기인,장기여표체재체pGEX-4T-1련접후전화대장간균유도표체획득중조 AK 단백(rAK),통과 pull-down 실험진일보증실 rAK 가여 rVP110발생결합。극씨원오하(Procambarus clarkia)체내중화실험결과현시, rAK대WSSV감염극씨원오하구유일정적중화작용,능연완오하적사망진정。령외,중국명대하재인공감염WSSV후,형광정량PCR검측결과현시, AK기인표체수평현저상조,18 h시체도봉치,연후하강지정상수평；매저물법검측결과동양현시,새세포중AK매활성재감염WSSV후발생현저상조。본연구지재위심입료해WSSV낭막단백VP110재WSSV감염숙주과정중적작용제공기출의거。
White spot syndrome virus (WSSV) is one of the most virulent viral pathogens of the shrimp-farming indus-try worldwide, causing high mortality and resulting in serious economic losses. The viral envelope proteins play very important roles in WSSV infection of shrimps. To investigate the interaction between WSSV envelope protein VP110 and gill cell proteins of the Chinese shrimp, Fenneropenaeus chinensis, a truncated VP110 protein containing the RGD motif was recombinantly expressed in Escherichia coli BL21(DE3) as a fusion protein with Trx/His/S-tag. Using a pull-down assay, two prominent protein bands with molecular weights of 40 kD and 42 kD were identified from gill cell proteins of F. chinensis using recombinant VP110 (rVP110), which were identified as arginine kinase (AK) andβ-actin by mass spectrometry (MS) analysis. The AK gene of F. chinensis was cloned and expressed as a fusion protein with GST-tag using the pGEX-4T-1 vector, and the binding interaction between the recombinant AK (rAK) and rVP110 was further confirmed by a pull-down assay. In an in vivo neutralization assay, rAK appeared to be able to partially block WSSV infection and delayed the death of WSSV-infected crayfish. In addition, following WSSV infection, AK mRNA level in the gills was upregulated compared with the control group, as assessed by real-time quantitative PCR. The AK enzyme activity in the gills was also upregulated. These results suggested that AK in F. chinensis plays a role in WSSV infection.
