scholarly journals Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

2022 ◽  
Author(s):  
Nguyen Quang Linh ◽  
Khanh Van Nguyen ◽  
Dung Quoc Tran ◽  
Van Khanh Tran Quang
Keyword(s):  
Recombinant Protein ◽  
Vibrio Parahaemolyticus ◽  
Expression Vector ◽  
Cloning And Expression ◽  
Economic Losses ◽  
Bacterial Disease ◽  
Parahaemolyticus Strain ◽  
E Coli ◽  
Whiteleg Shrimp ◽  
Acute Hepatopancreatic Necrosis Disease

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

scholarly journals Selection of Lactic Acid Bacteria (LAB) Antagonizing Vibrio parahaemolyticus: The Pathogen of Acute Hepatopancreatic Necrosis Disease (AHPND) in Whiteleg Shrimp (Penaeus vannamei)

Biology ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 91
Author(s):  
Linh Nguyen Thi Truc ◽  
Ai Trinh Ngoc ◽  
To Tran Thi Hong ◽  
Tuu Nguyen Thanh ◽  
Huong Huynh Kim ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Vibrio Parahaemolyticus ◽  
Positive Control ◽  
Control Treatment ◽  
Diffusion Method ◽  
Sequencing Analysis ◽  
Parahaemolyticus Strain ◽  
Whiteleg Shrimp ◽  
Acute Hepatopancreatic Necrosis Disease

Acute hepatopancreatic necrosis disease (AHPND) has recently emerged as a serious disease of cultured shrimp. A total of 19 lactic acid bacteria (LAB) strains isolated from shrimp samples were characterized based on morphological characteristics, biochemical tests, sequencing analysis, and their ability to antagonize Vibrio parahaemolyticus, which causes AHPND in whiteleg shrimp. Results from the agar well diffusion method indicated that 3 out of 19 isolated LAB strains showed the highest antagonizing ability against AHPND V. parahaemolyticus strain with an inhibition zone diameter ranging from 18 to 20 mm. Experiments where shrimps were given feed supplemented with these LAB strains and challenged with AHPND strain showed high survival rates (approximately 80.0%), which were not significantly different as compared to those recorded in the negative control treatment (86.6%), but significantly different to those recorded in the positive control treatment (40.6%) after 16 days of the experiment. However, the histological images of shrimp hepatopancreas indicated that the infection rate significantly reduced from 60.0% to 11.1% in shrimps fed with LAB-supplemented feeds and challenged with AHPND V. parahaemolyticus strain as compared to those in the positive control treatment. A polymerase chain reaction (PCR) and 16S rRNA gene sequencing confirmed the identification of LAB strain. These results can be applied in further experiments to investigate the ability of L. plantarum in preventing AHPND in intensively cultured whiteleg shrimp.

scholarly journals Specific T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese HIV Positive Individuals

2013 ◽  
Vol 2 (1) ◽  
pp. 6-12
Author(s):  
Bin Zhang ◽  
Wen-hui Lun ◽  
Xing-wang Li ◽  
Qi Wang ◽  
Jun Cheng ◽  
...  
Keyword(s):  
T Cell ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
T Cell Responses ◽  
Hiv Positive ◽  
E Coli ◽  
Cell Responses ◽  
Prokaryotic Expression Vector

Abstract Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity; the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94% in total bacterial protein of E. coli and the molecular weight is 31 kD; Western blot confirmed the recombinant proteins had high antigenicity; our in-house ELISpot-IFN-γ assay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

scholarly journals Cloning and expression of \(\textit{ pigI}\) gene in \(\textit{Escherichia coli}\) BL21 (DE3)

2021 ◽  
Vol 43 (3) ◽  
pp. 59-67
Author(s):  
Do Minh Trung ◽  
Do Hai Quynh ◽  
Nguyen Thuy Duong
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Biosynthetic Pathway ◽  
Condensation Reaction ◽  
Potential Method ◽  
Antimicrobial Activities ◽  
Cloning And Expression ◽  
E Coli ◽  
Recombinant Vector ◽  
His Tag

Prodigiosin (Pg), a secondary metabolite with anticancer and antimicrobial activities, can be produced in Serratia marcescens bacteria through the condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Among these, the MBC synthetic pathway is started by the conversion of L-proline to L-proline-AMP before this complex is covalently attached to PigG. This reaction is catalyzed by an L-prolyl-AMP ligase named PigI. Therefore, PigI protein plays an important role in the prodigiosin biosynthetic pathway. However, studies related to PigI protein have not been carried out in Vietnam yet. In this work, the pigI gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Sequence alignment results revealed that the obtained pigI gene is 99.7% identical to the four strains, CP027798, CP027796, CP021984 and CP003959. This recombinant vector pJET1.2/pigI was used to reamplify pigI, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing the recombinant vector pET22b/pigI was expressed in an auto-induced medium. The presence of PigI protein in the lysate was identified due to a 53 kDa band through Western Blot analysis using an anti-his-tag antibody. The results of our study provide a potential method for producing prodigiosin from recombinant protein in Vietnam.

scholarly journals ANTIBACTERIAL ACTIVITY OF HERBAL EXTRACTS AGAINST Vibrio parahaemolyticus CAUSING ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) IN WHITELEG SHRIMP (Litopenaeus vannamei) IN VITRO

2020 ◽  
Vol 1 (38) ◽  
pp. 82-88
Author(s):  
Nguyen Thi Nguyen ◽  
Nhi Thi Hong Nguyen
Keyword(s):  
Antibacterial Activity ◽  
Litopenaeus Vannamei ◽  
Vibrio Parahaemolyticus ◽  
Leaf Extract ◽  
Herbal Extracts ◽  
Physalis Angulata ◽  
Whiteleg Shrimp ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Muntingia Calabura

The antibacterial activity of herbal extracts to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in whiteleg shrimp (Litopenaeus vannamei) was studied and carried out in the School of Agriculture and Aquaculture, Tra Vinh University. The results showed that the inhibition zone of Muntingia calaburaleaf extract with ethanol 70% was 18.00 ± 0.00mm, Muntingia calabura leaf extract with ethanol 90% was 17.33 ± 0.58 mm and Muntingia calabura fresh leaf extract was 13.00 ± 0.00 mm. Minimum inhibitory concentration (MIC) of Muntingia calabura leaf extract with ethanol 70% and 90% were 5.120 mg/L, 10.240 mg/L. MIC value of Al lium cepa extract with ethanol 70% and 90% were 40.960 mg/L, Physalis angulata extract with ethanol 70% and 90% were 81.920 mg/L. The results showed that the antimicrobial activity was highest in the Muntingiacalabura leaf extract and lowest in Physalis angulata extract.

scholarly journals Bacterial expression of the recombinant functionally active N-terminal module of the B. taurus tyrosyl-tRNA synthetase

2017 ◽  
Vol 23 (2) ◽  
pp. 33-37
Author(s):  
V. Zayets ◽  
O. Tsuvariev ◽  
A. Kornelyuk ◽  
L. Kolomiyets ◽  
Keyword(s):  
Catalytic Activity ◽  
Nucleotide Sequence ◽  
Recombinant Protein ◽  
Expression Vector ◽  
Bos Taurus ◽  
Trna Synthetase ◽  
Bacterial Expression ◽  
E Coli ◽  
Physical And Chemical

The nucleotide sequence coding N-terminal module of Bos taurus tyrosyl-tRNA synthetase (mini TyrRS) was cloned into the bacterial expression vector pET23d(+). Bacterial expression of the recombinant protein mini TyrRS was performed in E. coli BL21 (DE3)pLysE cells with the use of the constructed vector pET-23d(+)39YRS for subsequent physical and chemical protein studies. The catalytic activity of the recombinant mini TyrRS has been studied in the aminoacylation reaction of homologous tRNATyr.

scholarly journals The infection of Vibrio parahaemolyticus in shrimp and human

2018 ◽  
Vol 1 (1) ◽  
pp. 44 ◽  
Author(s):  
Rian Ka Praja
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Animal Health ◽  
Alternative Therapy ◽  
Penaeus Monodon ◽  
Early Mortality ◽  
Economic Losses ◽  
Early Mortality Syndrome ◽  
Thermostable Direct Hemolysin ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Very High

<p class="15" align="justify"><em>Vibrio parahaemolyticus</em> is an aquatic zoonotic agent that can threaten human and aquaculture animal health. Humans can be infected by consuming contaminated raw seafood or wound-related infections. Generally infection of <em>V. parahemolyticus</em> is orally transmitted and causes gastroenteritis in humans while in aquaculture animals especially shrimp can cause Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) with a very high mortality rate and cause economic losses. Shrimp species susceptible to infection are <em>Litopenaeus vannamei, Penaeus monodon,</em> and <em>P. chinensis</em>. <em>V. parahaemolyticus</em> produces several toxins in human disease such as thermostable direct hemolysin (TDH), TDH-related haemolysin (TRH), and thermolabile hemolysin (TLH). Meanwhile, Photorabdus insect-related (Pir) toxins consisting of PirA<sup>vp</sup> and PirB<sup>vp</sup> are the toxins associated with AHPND in shrimp. The genes that encode the toxin are used as targets to diagnose <em>V. parahaemolyticus</em> pathogens molecularly. Until now the treatment of <em>V. parahaemolyticus</em> infection is using antibiotics and fluid therapy, but there were <em>V. parahaemolyticus</em> isolates from aquaculture that have been resistant to antibiotics so that the use of antibiotics in aquaculture must be controlled and the use of alternative therapy are very important to be developed to control <em>V. parahaemolyticus</em> infection.</p><p class="15" align="justify"> </p><p>Keywords: <em>V. parahaemolyticus</em>, zoonotic, gastroenteritis, Acute Hepatopancreatic Necrosis Disease (AHPND), Early Mortality Syndrome (EMS).</p>

scholarly journals Molecular characterization of acute hepatopancreatic necrosis disease causing Vibrio parahaemolyticus strains in cultured shrimp Penaeus monodon in south‐west farming region of Bangladesh

2018 ◽  
Vol 27 (1) ◽  
pp. 57-68 ◽  
Author(s):  
Md Mostavi Enan Eshik ◽  
Nusrat Jahan Punom ◽  
Mst Khadiza Begum ◽  
Tahsin Khan ◽  
Mihir Lal Saha ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Penaeus Monodon ◽  
16S Rrna Gene Sequencing ◽  
Molecular Techniques ◽  
Shrimp Farming ◽  
Economic Losses ◽  
Rrna Gene ◽  
South West ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Positive Results

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease caused by strains of Vibrio parahaemolyticus containing a unique virulent plasmid, responsible for substantial economic losses since 2009; caused up to 100% mortality in farmed shrimp Penaeus monodon. The purpose of this study was to isolate and identify the pathogenic strain of V. parahaemolyticus causing AHPND in cultured shrimp (Penaeus monodon) using classical and molecular techniques. Samples were collected from three different locations of south-west shrimp farming regions of Bangladesh viz. Sadar Upazilla of Satkhira; Mongla and Morrelganj under Bagerhat district. In this study, three selective media were used for primary isolation of V. parahaemolyticus. Among 46 primary isolates, 18 representative isolates were checked for the species-specific detection of V. parahaemolyticus using ldh primers and all of them were found to be positive. 16S rRNA gene sequencing were used to further confirm the isolates as V. parahaemolyticus. tdh primer was used to check human pathogenicity but all 18 isolates showed negative result. The isolates were further characterized to check their AHPND positivity using AP3 and AP4 primers. Ten isolates showed positive results for AP3 (55.56%) and 9 showed positive results for AP4 (50%) which indicated that the isolates were AHPND positive. This study also reported that all AHPND positive strains were resistant to the antibiotic gentamycin but sensitive to chloramphenicol, nalidixic acid, nitrofurantoin and tetracycline. The findings of this study will help the shrimp farmers and policy makers to take proper biosecurity measures to protect shrimps from AHPND and thereby sustain the shrimp production in Bangladesh. Dhaka Univ. J. Biol. Sci. 27(1): 57-68, 2018 (January)

Multifunctional Carbonized Nanogels to Treat Lethal Acute Hepatopancreatic Necrosis Disease

2021 ◽  
Author(s):  
Shao-Chieh Yen ◽  
Ju-Yi Mao ◽  
Hung-Yun Lin ◽  
Huai-Ting Huang ◽  
Scott G. Harroun ◽  
...  
Keyword(s):  
Functional Groups ◽  
Vibrio Parahaemolyticus ◽  
Feed Additives ◽  
Economic Losses ◽  
Multiple Drug ◽  
Loss Of Function ◽  
Bacterial Membranes ◽  
Resistant Strains ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
The Impact

Abstract Background: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. Methods: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 ℃ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. Results: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neuralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. Conclusion: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

scholarly journals Cloning and Expression of N-CFTX-1 Antigen from Chironex fleckeri in Escherichia coli and Determination of Immunogenicity in Mice

2021 ◽  
Author(s):  
Hossein JAFARI ◽  
Saeid TAMADONI JAHROMI ◽  
Jamil ZARGAN ◽  
Ehsan ZAMANI ◽  
Reza RANJBAR ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Elisa Test ◽  
Cloning And Expression ◽  
E Coli ◽  
Chironex Fleckeri ◽  
Jellyfish Species ◽  
Clone And Express ◽  
Bandar Abbas ◽  
The Persian Gulf ◽  
Jellyfish Venom

Background: Most jellyfish species are poisonous. Human victims of jellyfish sting each year are 120 million. Chironex fleckeri is a venomous box jellyfish that inflicts painful and potentially fatal stings to humans. The CfTX1 is one of the antigenic proteins of venom that is suggested to stimulate the immune system for treatment and vaccine. This study aimed to clone and express the CfTX-1 antigen in E. coli and then to determine the synthesis of related antibody in the mice. Methods: The study was performed in the Persian Gulf and Oman Sea Ecology Research Center, Bandar Abbas, Iran in autumn 2016. The synthetic CfTX-1 gene in PUC57 plasmid was purchased from Nedaye Fan Company. The 723 bp fragment of N-CfTX-1 was amplified by PCR, PUC57 plasmid containing CfTX-1 with BamHI SalI restriction enzyme sites were subcloned in pET28a [+] expression vector and transformed into E. coli BL21 (DE3). The CfTX-1 gene expression was induced by IPTG. Then antibody produced from the mice serum were isolated and confirmed by ELISA. After protein purification, resulted antigen was injected to mice in 4 repeats and then evaluated the rate of antibody in mice serum. Mice were challenged by the Carybdea alata. Results: The 726 bp of N-CfTX-1 were cloned in a vector of expression pET28a [+] and confirmed by PCR, sequencing and enzymatic analysis. Moreover, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Then the antibody was isolated from mice serum and confirmed by ELISA test. The results showed that immunized mice tolerated 50x LD501 of jellyfish venom. Conclusion: The CfTX-1 recombinant protein was able to protect the BALB/c mice against jellyfish venom. The produced protein can be used as a candidate for vaccine against jellyfish venom.

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