Yang Yuan-jie

Shandong Freshwater Fisheries Research Institute

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Jianlin Pan

Ministry of Agriculture and Rural Affairs

Zhiqiang Xu

South China Agricultural University

Quanping Lu

Shandong Freshwater Fisheries Research Institute

Guangli Cao

Soochow University

Yong Zou

Sun Yat-sen University

Renyu Xue

Linyi People's Hospital

Chengliang Gong

Soochow University

Yanfei Deng

Guangxi University

Jindi Ni

Minhang District Central Hospital

Jiachun Ge

Shandong Freshwater Fisheries Research Institute

Cooperative Institutions

Shandong Freshwater Fisheries Research Institute

Nanjing Agricultural University

Nanjing Normal University

Soochow University

Ministry of Agriculture and Rural Affairs

Chinese Academy of Sciences

Shandong Entry-Exit Inspection and Quarantine Bureau

Southwest University

Huazhong University of Science and Technology

Yangzhou University

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Evaluation of immune efficacy of GCRV vp6 DNA vaccine

Zhongguo Shuichan Kexue (2012)

Lin Liu XU Shi-ying Jinghui Li Yong Zou Jindi Ni

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Construction and immune efficacy studies on DNA vaccine with VP7 gene of grass carp reovirus

Xu Shiying Lin Liu Jinghui Li Yong Zou Jindi Ni

Double GCRV outer capsid protein VP7 genes(0.9 kb)and β-actin promoter(0.56 kb)from Megalobrama amblycephala were amplified and cloned into gene transfer vector pFastBacTM Dual to obtain recombinant pFastBac-β-VP71-VP72.Immune experiment and immunological effect were studied.The recombinant plasmids were divided into three groups based on doses:10,30,60 μg.30 μg pFastBacTM Dual group and control group were designed in addition.Immune efficacy was determined by means of transcriptions of VP7,antibody detection by indirect agglutination reaction and challenge experiment on the 14th,21st,28th,and 49th days after immunization.The results showed that pFastBac-β-VP71-VP72 was transformed into body cell,VP7 gene could be controlled for expression constantly by β-actin promoter,and transcription of VP7 was still in process on the 49th day.Antibody was produced and the titers reached their peak on the 21th day after immunization.The mortality rates were 0%,0% and 5% respectively in 10 μg,30 μg and 60 μg vaccine immunization groups,30% in pFastBacTM Dual group,and 100% in control group after GCRV injection.All results suggested that pFastBac-β-VP71-VP72 had good immuno-protective efficacy as DNA vaccine against GCRV.Therefore,the study provided important academic basis for research,development and application of the GCRV genetic vaccine.

Antibody titer

Grass carp

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Prokaryotic expression,purification and immune efficacy of VP6 protein of grass carp reovirus

JOURNAL OF FISHERIES OF CHINA (2012)

Lin Liu Shiying Xu Jinghui Li Yong Zou Jin-di Ni

In order to study the immuno-protective efficacy of GCRV-VP6 subunit vaccine,the recombinant expression vector pET28a(+)-VP6 was constructed by cloning vp6 gene of GCRV into the prokaryotic expression plasmid pET-28a(+).The results of SDS-PAGE and Western-blotting showed that the molecular mass of the recombinant VP6 protein was approximately 43 ku.The recombinant protein reached 20% of the total bacterial proteins,and was mainly in the form of insoluble bodies.The grass carp(60-120 g in body weight and 14-20 cm in body length)was immunized with 500 μg purified VP6 protein by Ni2+ affinity chromatography and antibody titers in the blood of fish were determined by means of indirect agglutination reaction on the 14th,21st,28th,49th and 70th days post-vaccination.The results showed that the specific antibody could be detected on the 14th day,reached peak on the 21st day and could be still detected on the 70th day after immunization.The fish in vaccinated group and control group were challenged with GCRV on 21st days after vaccine delivery and the relative survival rate in the vaccined group was 100%.These results provided important academic foundation for research and development of GCRV genetic vaccine.

Grass carp

10.3724/sp.j.1231.2012.27803

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Separation and determination on virulence genes of Vibrio cholerae in Fugu obscurus

Yang Yuan-jie

During the survey on diseases of Fugu obscurus from 2002 to 2004,two strains(J-1 and H-3) of pathogenic bacteria with typical symptoms were isolated from abdominal cavity and intestinal mucus of suffering Fugu obscurus.The two isolated strains were identified as non-01strains of Vibrio cholerae through biochemical test and serotype identification.It could be inferred that they are pathogens of bacterial enteritis of Fugu obscurus since the results of artificial infection are the same as the natural infection.In order to testify the classification of the isolated strains,the isolated strains were amplified by polymerase chain reaction(PCR) with two primers designed according to the sequence of Vibrio.The PCR products were DNA sequence of 451 bp.The result showed that systematic status between J-1,H-3 and N92001,N16961 was close.Enterotoxin with active subunit A named ctxA is one of the primary infection agents of Vibrio cholerae.While the pathogenicity is related with ctxA,the test of gene of enterotoxin is the most important step of diagnosis.Using N16961 strains as positive control,the same PCR DNA sequence of 400 bp was obtained in the PCR analysis.By sequencing and alignment,this 407 bp sequence of H-3 strains is identical with ctxA,which indicated that J-1 strains and H-3 strains were the pathogenic bacteria of F.obscurus.Bacterial diseases are common and frequent in process of aquaculture especially the intensive aquiculture.This paper shows introduces how to combine common method and PCR to isolate,identify and study bacteria. The pollution of non-01strains of V cholerae in aquatic product reflects in some way the degree of environmental pollution.Thus the study of pathogenicity is meaningful to evaluate if bacteria would cause disease and to limit its prevalence,so as to study and control the zoonosis.

Fugu

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Identification of Plesiomus shigelloides from hybridized prussian carp(Carassius auratus gibelio♀×Cyprinus carpio♂)

Yang Yuan-jie

A Gram negative pathogenic bacterium named strain YCS07-01 was isolated from the liver samples of moribund hybridized prussian carp(Carassius auratus gibelio♀×Cyprinus carpio♂) and was identified by physiological-biochemical characteristics,cultural traits,shape and phylongenetic analysis of 16S rDNA sequences.The isolated bacterium was oxidase positive,decomposed m-inositol and produced acid from the catabolism of d-glucose without gas,and was unable to ferment sucrose,mannitol and rhamnose.The tests for β-galactosidase,arginine dihydrolase,lysine decarboxylase and ornithine decarboxylase were positive.Negative staining under electron microscope revealed that the isolated bacterium was short straight rods with rounded ends in size of 0.8~1.0 μm×3.0 μm,and with four flagella without gemma and capsule.A phylogenetic tree was constructed based on 16S rDNA sequences of strain YCS07-01(GenBank accession number FJ375179) and other relative bacteria species in the GenBank database.The phylogenetic tree revealed that strain YCS07-01 shared high similarity of 99% to that of Plesiomus shigelloides and formed a cluster with strain ATCC14029T(accession number X74688).The isolated bacterium strain YCS07-01 was confirmed as Plesiomus shigelloides by cultural traits,shape,physiological-biochemical characteristic identification and phylongenetic analysis.The drug sensitive tests demonstrated that the bacterium was resistant to carbenicillin and vancomycin,but highly sensitive to amikacin,levofloxacin,ceftazidime,sulfamethoxazole compound etc.

Lecithinase

Crucian carp

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Evaluation of immune efficacy of GCRV vp6 DNA vaccine

Journal of Fishery Sciences of China (2013)

Lin Liu Shiying Xu Jinghui Li Yong Zou Jindi Ni

10.3724/sp.j.1118.2012.00841

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Comparative transcriptome sequencing of the hepatopancreas reveals differentially expressed genes in the precocious juvenile Chinese mitten crab,Eriocheir sinensis(Crustacea: Decapoda)

Aquaculture Research (2016)

Yu Xu Xuguang Li Yanfei Deng Quanping Lu Yang Yuan-jie

Overnutrition was considered to be the main cause of the precocity in the Chinese mitten crab. Hepatopancreas plays a key role in nutrition metabolism of the crustaceans. However, gene regulation and expression profiles of the hepatopancreas for the precocious crabs still remain ill-defined. Here through de novo sequencing coupled with RNA-Seq (Quantification) analysis, a number of differentially expressed genes (DEGs) in the precocious juvenile crabs were identified. The pooled total RNA from hepatopancreas of the precocious juvenile crabs, normal juvenile crabs and normal matured crabs was used to obtain a reference transcriptome. De novo assembly of the transcriptome revealed 55 561 unigenes with an average length of 1153 bp. Another three cDNA libraries were constructed with the same samples and sequenced separately with the RNA-Seq analysis. A total of 996 DEGs between the precocious and normal juvenile crabs were identified, of which 508 DEGs were upregulated in the precocious juvenile crabs. KEGG enrichment analysis revealed that most enriched DEGs (162, 23.28%) were involved in the metabolic pathways. The expression profiles of 15 DEGs, which includes chitinase, alkaline phosphatase, insulin receptor, juvenile hormone esterase and signal transducer and activator of transcription, were determined by qRT-PCR to confirm the accuracy of the RNA-Seq. These DEGs had already been suggested to be potentially related to the growth and development in crustaceans. Our results, including the assembled transcriptome and the identified DEGs in the precocious juvenile crabs, provide us new resources in the understanding of the functions of the hepatopancreas in the precocity of the Chinese mitten crab.

Hepatopancreas

Chinese mitten crab

KEGG

10.1111/are.13189

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Isolation of the Pathogens and Histopathological Study on Ulcer Disease of Loach

Journal of Jimei University Natural Science (2004)

Yang Yuan-jie

Four pathogenic strains of bacteria,i.e.,02-7-01?02-7-02?02-7-03?02-7-04respectively,were isolated from the loaches affected with ulcer.Healthy loaches artificially affected with the pathogenic strain of bacteria 02-7-04 show strong infectious ability,and the similar symptoms as the naturally affected loaches.It primarily defined the strain 02-07-04 was the pathogenic strain of bacteria.Based on the morphologic characteristics,culture characteristics and biochemical reactions,the strain 02-7-04 is approved to be a kind of vibrio,V.vulnificus.The histological and pathological observations of the ulcer showed that muscle cells were necrotic and a lot of inflammatory cells were soaking;subcutaneous muscle tissues in the ulcer area were swollen and soaked with inflammatory cells;and the pathogenic bacterium invented from the intestinal tract caused the putrescence of intestinal mucous membrane soaked a large amount of inflammatory cells.The tests on the drug allergy indicated that the pathogenic bacterium strain 02-7-04 was sensitive to ciprofloxacin,gentamycin,norfloxacin,cefuroxime,cefoperazone,ceftazidime,and ceftriaxone,but was insensitive to ampicillin,piperacillin and cephazoline.

Pathogenic bacteria

Cefoperazone

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Molecular cloning, characterization and expression analysis of two juvenile hormone esterase-like carboxylesterase cDNAs in Chinese mitten crab, Eriocheir sinensis Part B Biochemistry & molecular biology

Comparative Biochemistry and Physiology (2017)

Yu Xu Muzi Zhao Yanfei Deng Yang Yuan-jie Xuguang Li

Hepatopancreas

Chinese mitten crab

Eyestalk

Carboxylesterase