| Crustaceans in aquaculture occupy a very important position and shrimp aquaculture has increased rapidly from an insignificant base in the early1980s,it has brought considerable economic benefits for coastal areas. But the shrimp diseases also attack the shrimp aquaculture and economy with the increase of scale. In recent years, shrimp immunological and pathology study has been progressed rapidly. The immune-related genes which have been cloned continuously were studied deeply.1. Anti-lipopolysaccharide factors (ALFs) play crucial roles in innate immunity of crustaceans to recognize and eliminate pathogens efficiently. The deep research about ALF will benefit to the control of shrimp diseases. In the present paper, an ALF gene from shrimp Litopenaeus vannamei was cloned and the recombinant expression of ALF was studied, including the following several aspects.The primers was designed according to ALF-like gene in NCBI Bank, and this gene from shrimp Litopenaeus vannamei was cloned by rapid amplification of cDNA ends (RACE) approach and designated as ALF-AVK according to the AVK (Ala-Val-Lys) sequence containing in its encoded protein. The ALF-AVK full-length cDNA is705bp, consisting of a43bp5’ terminal untranslated region (UTR), a293bp3’ UTR with a poly (A) tail and a369bp open reading frame (ORF). ALF-AVK encodes a protein of122amino acids with a predical molecular mass of13.7kDa and a theoretical isoelectric point of9.95.2. The SYBR Green qPCR analysis was employed to study the ALF-AVK mRNA expression in the tissues of intestine, gill, hemocyte, heart, muscle, and hepatopancreas of healthy shrimps with β-actin as internal control. The highest mRNA expression level of ALF-AVK was observed in hemocyte, while moderate expression levels were observed in gill and heart, and very low in muscle, intestine and hepatopancreas. Hemocytes which are the main defense tissue in the shrimp were chosen to evaluate the variation of ALF-AVK expression post white spot syndrome virus (WSSV) and Vibrio anguillarum challenge by quantitative real-time PCR (qPCR). A considerable up-regulation of ALF-AVK in hemocytes within6h post WSSV challenge was observed and exhibited a response of acute-phase; after a decrease to original level at24h, the expression of ALF-AVK increased until96h post challenge. In the V. anguillarum challenge group, the expression of ALF-AVK reached peak at12h and decreased slowly until96h.3. With the ALF-AVK was expressed in PET-30a plasmid and then induced by IPTG screening method. The purified recombinant protein rALF-AVK was purified by His-tag affinity column and was used as antigen to prepare antiserum in Balb/C mouse. We got a specific band in the expected position in the hemocytes of WSSV and V. anguillarum challenged L. vannamei at molecular weights of14kDa by Western Blot detection. The polyclonal antibodies against rALF-AVK could react specifically with a protein band at molecular weights of21kDa.4. To determine whether the antibacterial activity of ALF-AVK requires the ability to bind to the bacterial cells, bacterial-protein pull-down assay and enzyme-linked immunosorbent assay (ELISA) was carried out. The ELISA result showed that the experiment groups exhibited higher ELISA value with the amounts of purified rALF-AVK increased. In bacterial-protein pull-down assay, Coomassie Brilliant Blue G250SDS-PAGE resolved samples were interpretable due to the presence of rALF-AVK observed in21kDa and no band in negative control group. To identify whether ALF play roles in involving V. anguillarum and WSSV infection, the neutralization experiment was carried out in shrimp. The result showed that the mortality levels in groups injected with V. anguillarum pre-incubated with purified rALF-AVK were lower than that of control groups, which reach to100%at17th post challenge, but there was no effect in WSSV challenge group.5. Using the antiserum of rALF-AVK as the primary antibody, paraffin method and immunohistochemistry were employed to observe distribution of ALF inhemocyte, gill, heart, intestine, and hepatopancreas of L. vannamei. Immunohistochemistry results showed that the ALF existed mainly in hemocyte and positive signals were also found in blood vessel of gill, lacunaes among myocardial bundles of heart, hepatopancreatic tubules and blood sinus in connective tissues of intestine. In conclusion, hemocyanin was mostly from the hemolymph rather than fundamental tissue, and widely distuibuted in various tissues of L. vannamei. The positive signal in the organizationg of shrimps calleaged by V. anguillarum will be significantly increased compare with untreated shrimps. These results indicated that ALF was involved in the immune defense against V. anguillarum. |
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