Z.H. Wu

Cloning and expression of heat shock protein 70 gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850) responding to bacterial challenge

The cDNA of pearl oyster Pinctada fucata Hsp70 (designated PFHsp70) was cloned by EST and rapid amplification of cDNA ends (RACE) techniques. The full length of PFHsp70 cDNA was 2376 bp, consisting of a 5-terminal untranslated region (UTR) of 89 bp, a 3 terminal UTR of 328 bp, and an open reading frame (ORF) of 1959 bp encoding a polypeptide of 652 amino acids with a theoretical molecular weight of 71.42 kDa and an estimated isoelectric point of 5.18. BLAST analysis revealed that the PFHsp70 gene shared high similarity with other Hsp70 genes. PFHsp70 contained all the three classical Hsp70 family signatures. The results indicated that the PFHsp70 was a member of the heat shock protein 70 family. Fluorescent real-time quantitative RT-PCR was used to examine the expression of PFHsp70 gene in haemocytes of P. fucata after the challenge of bacteria Vibrio alginolyticus. There was a clear time-dependent expression pattern of PFHsp70 after bacterial challenge, and the mRNA expression reached a maximum level at 4 h post-challenge, which returned to control level after 32 h. The up-regulated mRNA expression of PFHsp70 in P. fucata after bacterial challenge indicates that the Hsp70 gene is inducible and involved in immune response.

Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish

Aims:u2002 The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish.

Cloning and expression of gene encoding the thermostable direct hemolysin from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis of crimson snapper (Lutjanus erythopterus)

Aims:u2002 The main aims of this study were to clone and express complete open reading frame (ORF) of thermostable direct haemolysin gene (tdh) from Vibrio alginolyticus strain HY9901 in Escherichia coli, and further evaluate the virulence of expressed TDH on mouse and crimson snapper.

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish.

Aims:u2002 The purpose of this study was to establish a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish.

Immunoproteomic analysis and identification of novel immunogenic proteins from Vibrio harveyi.

Aims:u2002 The main aim of this study was to screen novel immunogenic proteins of Vibrio harveyi, which could be vaccine candidates.

Protection of red snapper (Lutjanus sanguineus) against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene

Aims:u2002 The main aims of this study were to construct a DNA vaccine containing flagellin flaA gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of pcDNA‐flaA as a DNA vaccine candidate for red snapper (Lutjanus sanguineus).

Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901.

Aims:u2002 The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti‐FlaA polyclonal antibody for future pathogen or vaccine study.

Cloning and expression of an actin gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850)

An actin gene (designated pfact1) of pearl oyster, Pinctada fucata, was cloned from haemocytes by the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full length of Pfact1 cDNA was 1608xa0bp in length, having a 5 untranslated region (UTR) of 82xa0bp, a 3 UTR of 395xa0bp, and an open reading frame (ORF) of 1131xa0bp encoding a polypeptide of 376 amino acids with a predicted molecular weight of 41.76xa0kDa and an estimated isoelectric point of 5.29. Sequence analysis revealed that Pfact1 shared high similarity with other actins and was more closely related to vertebrate cytoplastic actins than muscle types. Phylogenetic analysis indicated that molluscan actins could also be generally grouped into two classes: muscle type and cytoplasmic type, although both are similar to vertebrate cytoplastic actins. Fluorescent real-time quantitative RT-PCR was used to examine the expression level of Pfact1 in haemocytes of P. fucata after the challenge of Vibrio alginolyticus, and results showed that Pfact1 exhibited stable expression in all time points, indicating that Pfact1 could be a suitable internal control for gene expression analysis in haemocytes of P. fucata.

Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping

To perform a systematic evaluation of the applicability, validity and reliability of the long PCR‐RFLP of 16S‐ITS‐23S rRNA genes for bacterial genotyping using both sequences retrieved from public genome databases and the experimental data obtained on bacterial cultures.

Whole-Genome Sequences of an Aerobic Anoxygenic Phototroph, Blastomonas sp. Strain AAP53, Isolated from a Freshwater Desert Lake in Inner Mongolia, China

ABSTRACT Blastomonas is a strictly aerobic bacteriochlorophyll a-producing genus within the alpha-4 Proteobacteria. Here we report the first genome sequence from this genus. The draft genome of Blastomonas sp. strain AAP53 contains a split photosynthesis gene cluster and two gene clusters encoding a flagellar system. Genes for the autotrophic CO2 fixation pathway are absent.