Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame en...Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame encoding 132 amino acids was cloned. Its deduced amino acid sequence contained the conserved functional domain of ALFs, the LPS binding domain （LBD）. its genotnic sequence consisted of three exons and four introns, nLvALF2 was mainly expressed in the Oka organ and gills of shrimps. The transcriptional level of nLw4LF2 increased significantly after white spot syndrome virus （WSSV） infection, suggesting its important roles in protecting shrimps from WSSV. Single nucleotide polymorphisms （SNPs） were found in the genomic sequence of nLvALF2, of which 38 were analyzed for associations with the susceptibility/resistance of shrimps to WSSV. The loci g.2422 A〉G, g.2466 T〉C, and g.2529 G〉A were significantly associated with the resistance to WSSV （P〈0.05）. These SNP loci could be developed as markers for selection of WSSV-resistant varieties ofLitopenaeus vannamei.展开更多
Anti-lipopolysaccharide factors （ALFs） are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In th...Anti-lipopolysaccharide factors （ALFs） are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Eseherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, （NH4）2SO4 and KH2PO4, were further optimized via the central composite design （CCD）. Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside （IPTG） concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L （NH4）2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7~C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% （without optimization） to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 105% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.展开更多
Anti-lipopolysaccharide factors(ALFs)exhibit a potent antimicrobial activity against a broad range of bacteria,filamentous fungi,and viruses.In previous reports,seven groups of ALFs(groups A–G)were identified in pena...Anti-lipopolysaccharide factors(ALFs)exhibit a potent antimicrobial activity against a broad range of bacteria,filamentous fungi,and viruses.In previous reports,seven groups of ALFs(groups A–G)were identified in penaeid shrimp.Among them,group D showed negative net charges and weak antimicrobial activity.Whether this group has antiviral function is not clear.In this study,the ALF sequences of penaeid shrimp were analyzed,and eight groups of ALF family(groups A–H)were identified.The four ALFs including MjALF-C2,MjALF-D1,MjALF-D2,and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli,and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus(WSSV)infection.Results showed that the expression of Vp28(WSSV envelope protein)decreased significantly in the MjALF-D2-injected shrimp only.Therefore,MjALF-D2 was chosen for further study.Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV.The WSSV replication was detected in RNA,genomic DNA,and protein levels using VP28 and Ie1(immediate-early gene of WSSV)as indicators in MjALF-D2-injected shrimp following WSSV infection.Results showed that WSSV replication was significantly inhibited compared with that in the rTRX-or PBS-injected control groups.After knockdown of MjAlf-D2 in shrimp by RNA interference,the WSSV replication increased significantly in the shrimp.All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity,and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.展开更多
Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,...Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease.In this study,we identified single nucleotide polymorphisms(SNPs)in the Litopenaeus vannamei anti-lipopolysaccharide factor 1(nLvALF1)and penaeidin 2-1(PEN2-1)genes,and we analyzed the associations between these SNPs and resistance/susceptibility to V.parahaemolyticus infection in the Pacific white shrimp.Postlarvae(PL20)shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days.DNA was extracted from susceptible(died within 6 days)and resistant(survived the challenge)shrimp(45 individuals/group)and used for PCR amplifications of nLvALF1(397 bp)and PEN2-1(637 bp)gene fragments.PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms.Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments,respectively.Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1,respectively,were associated with resistance/susceptibility to V.parahaemolyticus infection(P<0.05).The SNPs in the candidate genes identified here are potential DNA markers for breeding V.parahaemolyticus-resistant Pacific white shrimp in the study population;however,further validation will be required if these SNPs are to be used across populations.展开更多
目的研究鲎抗内毒素因子模拟肽4(m ode ling peptide 4 from the L im u lus an ti-lipopo lysaccharide factor,M 4)体外中和内毒素的活性。方法研究以对内毒素高亲和力的多粘菌素B(po lym yx in B,PM B)为对照,M 4设80、40、20、10、5...目的研究鲎抗内毒素因子模拟肽4(m ode ling peptide 4 from the L im u lus an ti-lipopo lysaccharide factor,M 4)体外中和内毒素的活性。方法研究以对内毒素高亲和力的多粘菌素B(po lym yx in B,PM B)为对照,M 4设80、40、20、10、5μm o l/L浓度,结合内毒素的活性以生物传感技术分析;中和内毒素的能力以鲎试验动态浊度法测定;对LPS刺激THP-1细胞释放TNF-α的影响采用EL ISA试剂盒定量观察。结果M 4具有较好的内毒素亲和力和中和活性,在5、10、20、40和80μm o l/L浓度时对内毒素2EU/m l(1EU约等于100pg)的中和率分别为43.44%、47.76%、63.12%、81.54%和84.54%,呈明显的浓度梯度效应,20μm o l/L以上浓度时与PM B作用相当。对THP-1细胞释放TNF-α的影响结果显示,M 4可明显抑制LPS刺激的TNF-α释放,但M 4本身具有诱导THP-1细胞释放TNF-α的作用。结论M 4具有较强的中和内毒素活性,值得进一步深入研究。展开更多
基金Supported by the National High Technology Research and Development Program of China(863 Program)(Nos.2012AA10A404,2012AA092205)the General Program of National Natural Science Foundation of China(Nos.31272683,31072203)to Dr.LI Fuhua
文摘Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame encoding 132 amino acids was cloned. Its deduced amino acid sequence contained the conserved functional domain of ALFs, the LPS binding domain （LBD）. its genotnic sequence consisted of three exons and four introns, nLvALF2 was mainly expressed in the Oka organ and gills of shrimps. The transcriptional level of nLw4LF2 increased significantly after white spot syndrome virus （WSSV） infection, suggesting its important roles in protecting shrimps from WSSV. Single nucleotide polymorphisms （SNPs） were found in the genomic sequence of nLvALF2, of which 38 were analyzed for associations with the susceptibility/resistance of shrimps to WSSV. The loci g.2422 A〉G, g.2466 T〉C, and g.2529 G〉A were significantly associated with the resistance to WSSV （P〈0.05）. These SNP loci could be developed as markers for selection of WSSV-resistant varieties ofLitopenaeus vannamei.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2006AA100311)
文摘Anti-lipopolysaccharide factors （ALFs） are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Eseherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, （NH4）2SO4 and KH2PO4, were further optimized via the central composite design （CCD）. Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside （IPTG） concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L （NH4）2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7~C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% （without optimization） to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 105% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.
基金supported by grants from the National Natural Science Foundation of China(grant nos.31630084 and 31930112)the National Key Research and Development Program of China(grant no.2018YFD0900502).
文摘Anti-lipopolysaccharide factors(ALFs)exhibit a potent antimicrobial activity against a broad range of bacteria,filamentous fungi,and viruses.In previous reports,seven groups of ALFs(groups A–G)were identified in penaeid shrimp.Among them,group D showed negative net charges and weak antimicrobial activity.Whether this group has antiviral function is not clear.In this study,the ALF sequences of penaeid shrimp were analyzed,and eight groups of ALF family(groups A–H)were identified.The four ALFs including MjALF-C2,MjALF-D1,MjALF-D2,and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli,and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus(WSSV)infection.Results showed that the expression of Vp28(WSSV envelope protein)decreased significantly in the MjALF-D2-injected shrimp only.Therefore,MjALF-D2 was chosen for further study.Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV.The WSSV replication was detected in RNA,genomic DNA,and protein levels using VP28 and Ie1(immediate-early gene of WSSV)as indicators in MjALF-D2-injected shrimp following WSSV infection.Results showed that WSSV replication was significantly inhibited compared with that in the rTRX-or PBS-injected control groups.After knockdown of MjAlf-D2 in shrimp by RNA interference,the WSSV replication increased significantly in the shrimp.All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity,and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.
基金Funding for this research was provided by the National Science and Technology Development Agency,Ministry of Higher Education,Science,Research and Innovation.
文摘Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease.In this study,we identified single nucleotide polymorphisms(SNPs)in the Litopenaeus vannamei anti-lipopolysaccharide factor 1(nLvALF1)and penaeidin 2-1(PEN2-1)genes,and we analyzed the associations between these SNPs and resistance/susceptibility to V.parahaemolyticus infection in the Pacific white shrimp.Postlarvae(PL20)shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days.DNA was extracted from susceptible(died within 6 days)and resistant(survived the challenge)shrimp(45 individuals/group)and used for PCR amplifications of nLvALF1(397 bp)and PEN2-1(637 bp)gene fragments.PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms.Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments,respectively.Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1,respectively,were associated with resistance/susceptibility to V.parahaemolyticus infection(P<0.05).The SNPs in the candidate genes identified here are potential DNA markers for breeding V.parahaemolyticus-resistant Pacific white shrimp in the study population;however,further validation will be required if these SNPs are to be used across populations.
文摘目的研究鲎抗内毒素因子模拟肽4(m ode ling peptide 4 from the L im u lus an ti-lipopo lysaccharide factor,M 4)体外中和内毒素的活性。方法研究以对内毒素高亲和力的多粘菌素B(po lym yx in B,PM B)为对照,M 4设80、40、20、10、5μm o l/L浓度,结合内毒素的活性以生物传感技术分析;中和内毒素的能力以鲎试验动态浊度法测定;对LPS刺激THP-1细胞释放TNF-α的影响采用EL ISA试剂盒定量观察。结果M 4具有较好的内毒素亲和力和中和活性,在5、10、20、40和80μm o l/L浓度时对内毒素2EU/m l(1EU约等于100pg)的中和率分别为43.44%、47.76%、63.12%、81.54%和84.54%,呈明显的浓度梯度效应,20μm o l/L以上浓度时与PM B作用相当。对THP-1细胞释放TNF-α的影响结果显示,M 4可明显抑制LPS刺激的TNF-α释放,但M 4本身具有诱导THP-1细胞释放TNF-α的作用。结论M 4具有较强的中和内毒素活性,值得进一步深入研究。
基金Supported by the National Natural Science Foundation of China(31570176,31602198)the Natural Science Foundation of Jiangsu Province(BK20151545)Project for Aquaculture in Jiangsu Province(D2015-13,Y2016-28)
文摘为了研究抗脂多糖因子ALFs在日本沼虾先天性免疫中的功能作用,研究从日本沼虾中克隆了2种抗脂多糖因子Mn ALF1、Mn ALF2。Mn ALF1 c DNA全长1008 bp,编码121个氨基酸;Mn ALF2 c DNA全长836 bp,编码124个氨基酸。这2种氨基酸均包含有一个信号肽序列和一个LPS结合位点,并且在结合位点的两端(N-端和C-端)都有2个保守的半胱氨酸残基。这2种Mn ALFs与之前发现的甲壳动物的ALFs是非常相似的。q RT-PCR结果显示Mn ALFs在所有被检测的组织中均有表达。其中Mn ALF1主要在心脏和小肠内表达,而Mn ALF2则主要在血细胞和肝胰脏中表达。在用嗜水气单胞菌刺激之后发现2种Mn ALFs在心脏、小肠、血细胞、肝胰脏中都呈现出明显的时间依赖表达模式(Mn ALF1在刺激之后呈现出先减少后增加的趋势,之后分别在不同组织的不同时间点达到最大值;然而,对于Mn ALF2,在心脏和小肠中先减少后增加,在血细胞和肝胰脏中呈现出先增加后减少,最后都在24h达到最大值)。结果提示这2种Mn ALF具有不同的组织特异性,并且在细菌侵染的免疫防御中起着重要的保护作用。
