欧文氏弧菌毒力蛋白PirAB的原核表达与纯化
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摘要
对虾肝胰腺坏死病的爆发造成了对虾养殖产业的严重亏损。从上海地区凡纳滨对虾中分离出1株欧文氏弧菌(Vibrio owensii SH-14),该菌株可导致凡纳滨对虾死亡,且死虾出现对虾肝胰腺坏死病的典型症状。经PCR扩增欧文氏弧菌毒力蛋白PirA与PirB对应基因序列,并将其连接到表达质粒pET-21b(pirA)和pGEX-4t-1(pirB)。通过优化诱导表达和亲和层析纯化条件,最终获得大量高纯度的目的蛋白。经冷冻干燥保存为后期抗体合成以及进一步毒力效应研究提供参考。
The fulmination of acute hepatopancreatic necrosis disease(AHPND) creates severe loss to shrimp farming industry. An AHPND causing-strain Vibrio owensii SH-14 was isolated from white prawn(Litopenaeus vannamei) in Shanghai and confirmed to create the death of the shrimp and the shrimp appeared typical symptoms of AHPND. In this study, both pirA and pirB corresponding genes of virulent proteins in the V. owensii SH-14 were amplified by PCR and linked to plasmid vectors of pET-21 b and pGEX-4 t-1 respectively, and expressed in E. coli. Finally a large scale of the PirAB objective proteins were highly purified with affinity chromatography and frozen-dried and conserved for later systhesis of antibody, and laid a foundation for further study on the virulent effect.
The fulmination of acute hepatopancreatic necrosis disease(AHPND) creates severe loss to shrimp farming industry. An AHPND causing-strain Vibrio owensii SH-14 was isolated from white prawn(Litopenaeus vannamei) in Shanghai and confirmed to create the death of the shrimp and the shrimp appeared typical symptoms of AHPND. In this study, both pirA and pirB corresponding genes of virulent proteins in the V. owensii SH-14 were amplified by PCR and linked to plasmid vectors of pET-21 b and pGEX-4 t-1 respectively, and expressed in E. coli. Finally a large scale of the PirAB objective proteins were highly purified with affinity chromatography and frozen-dried and conserved for later systhesis of antibody, and laid a foundation for further study on the virulent effect.
引文
[1] Lightner DV, Redman RM, Pantoja CR, et al.Early mortality syndrome affects shrimp in Asia[J]. Glob Aquacult Advocate,2012,15(1):40-42.
[2] FAO Fisheries and Aquaculture: Report of the FAO/MARD technical workshop on early mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS) of cultured shrimp.Available online:www.fao.org/docrep/018/i3422e/i3422e.pdf (accessed on 31 October 2013).
[3] Tran L, Nunan L, Redman R M, et al. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J]. Diseases of aquatic organisms, 2013,(105): 45-55.
[4] Flegel TW. Historic emergence, impact and current status of shrimp pathogens in Asia[J]. Journal of invertebrate pathology, 2012, 110(2): 166-173.
[5] 唐小千,徐洪森,战文斌,等.对虾急性肝胰腺坏死综合症研究进展[J].海洋湖沼通报, 2016,(2):90-93.
[6] 文国樑, 曹煜成, 徐煜,等. 养殖对虾肝胰腺坏死综合症研究进展[J]. 广东农业科学, 2015, 42(11):118-123.
[7] 刘群, 黄倢, 杨昊霖,等. 疑患EMS/AHPNS对虾中检出黄头病毒的一种新株型[J]. 海洋与湖沼, 2014, 45(4):703-709.
[8] GomezGil B, SotoRodríguez S, Lozano R, et al. Draft genome sequence of Vibrio parahaemolyticus strain M0605, which causes severe mortalities of shrimps in Mexico[J]. Genome announcements, 2014, 2(2):e00055-14.
[9] Lee CT,Chen IT, Yang YT, et al. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J].Proc Natl Acad Sci,2015,112:10798-10803.
[10] Han JE, Tang KFJ, Tran LH, et al. Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp[J]. Diseases of aquatic organisms, 2015, 113(1):33-40.
[11] Liu L, Jiang J, Yu Y, et al. Rapid diagnosis of Vibrio owensii responsible for shrimp acute hepatopancreatic necrosis disease with isothermal recombinase polymerase amplification assay[J].Mol Cell Probes,2017,33(7):4-7.
[12] Xiao J, Liu L,Ke Y,et al.Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species[J].Scientific Reports,2017,7(2):42177.
[13] Liu L,Xiao J,Xia X, et al.Draft Genome Sequence of Vibrio owensii Strain SH-14, Which Causes Shrimp Acute Hepatopancreatic Necrosis Disease[J].Genome Announc,2015, 3(6):e01395-15.
[14] Liu L,Xiao J,Zhang M,et al.A Vibrio owensii strain as the causitive agents of AHPND in cultured Shrimp,Litopenaeus vannamei[J].Journal of Invertebrate Pathology,2018,153:156-164.
[2] FAO Fisheries and Aquaculture: Report of the FAO/MARD technical workshop on early mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS) of cultured shrimp.Available online:www.fao.org/docrep/018/i3422e/i3422e.pdf (accessed on 31 October 2013).
[3] Tran L, Nunan L, Redman R M, et al. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J]. Diseases of aquatic organisms, 2013,(105): 45-55.
[4] Flegel TW. Historic emergence, impact and current status of shrimp pathogens in Asia[J]. Journal of invertebrate pathology, 2012, 110(2): 166-173.
[5] 唐小千,徐洪森,战文斌,等.对虾急性肝胰腺坏死综合症研究进展[J].海洋湖沼通报, 2016,(2):90-93.
[6] 文国樑, 曹煜成, 徐煜,等. 养殖对虾肝胰腺坏死综合症研究进展[J]. 广东农业科学, 2015, 42(11):118-123.
[7] 刘群, 黄倢, 杨昊霖,等. 疑患EMS/AHPNS对虾中检出黄头病毒的一种新株型[J]. 海洋与湖沼, 2014, 45(4):703-709.
[8] GomezGil B, SotoRodríguez S, Lozano R, et al. Draft genome sequence of Vibrio parahaemolyticus strain M0605, which causes severe mortalities of shrimps in Mexico[J]. Genome announcements, 2014, 2(2):e00055-14.
[9] Lee CT,Chen IT, Yang YT, et al. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J].Proc Natl Acad Sci,2015,112:10798-10803.
[10] Han JE, Tang KFJ, Tran LH, et al. Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp[J]. Diseases of aquatic organisms, 2015, 113(1):33-40.
[11] Liu L, Jiang J, Yu Y, et al. Rapid diagnosis of Vibrio owensii responsible for shrimp acute hepatopancreatic necrosis disease with isothermal recombinase polymerase amplification assay[J].Mol Cell Probes,2017,33(7):4-7.
[12] Xiao J, Liu L,Ke Y,et al.Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pirAB-Tn903 are prevalent in various Vibrio species[J].Scientific Reports,2017,7(2):42177.
[13] Liu L,Xiao J,Xia X, et al.Draft Genome Sequence of Vibrio owensii Strain SH-14, Which Causes Shrimp Acute Hepatopancreatic Necrosis Disease[J].Genome Announc,2015, 3(6):e01395-15.
[14] Liu L,Xiao J,Zhang M,et al.A Vibrio owensii strain as the causitive agents of AHPND in cultured Shrimp,Litopenaeus vannamei[J].Journal of Invertebrate Pathology,2018,153:156-164.