The Expression And Functional Identification Of Astakine Of Litopenaeus Vannamei

Astakine was a cytokine that promotes cell differentiation and proliferation of hemocytes, which played important roles in crustacean immune responses. Studies showed that F₁F₀-ATP synthaseβsubunit participated in the cell differentiation as astakine receptor in Pacifastacus leniusculus, while in our previous studies F₁F₀-ATP synthaseβsubunit (named BP53) played a role in White Spot Syndrome Virus's (WSSV) infection as a candidate receptor. That is to say, BP53 was used as receptor by both WSSV and astakine. Based on the previous studies above, we speculate WSSV may compete BP53 with astakine, causing some interference in cell differentiation. The knowledge of astakine will help us better understanding the molecular mechanism of WSSV infection.Five parts included in this research. (1)The resulting full-length gene of astakine(lvast) was abtained by rapid amplification of cDNA ends (RACE) PCR. (2) The preparation of polyclonal antibody against LVAST by using the recombinant astakine. (3) Analysis of the interaction among BP53, LVAST and VP37 by ELISA, competitive ELISA and Dynabeads. (4) Analysis of recombinant LVAST's function in shrimp litopenaeus vannamei against WSSV by in vivo neutralization assay. (5) Effect on viral replication of astakine by RNAi and Real-time PCR assay.The main results in details as followed:(1) The resulting full-length gene of lvast was abtained by rapid amplification of cDNA ends (RACE) PCR. By rapid amplification of cDNA ends (RACE) PCR, the resulting full-length gene of lvast was abtained, which consisted of 1486 bp with a 372 bp ORF, encoding 124 amino acids with the predicted molecular mass 13.3 kDa. The deduced protein contained a prokineticin (PK) domain. A homology search against GenBank using BLAST, the sequence showed 87 % and 56 % similarity with astakine of Penaeus monodon and Pacifastacus leniusculus separately, and less than 36 % similarity with astakine of vertebrates. (2) The preparation of polyclonal antibody against LVAST by using the recombinant astakine. The lvast gene was amplified by PCR and cloned into vector pBAD/gⅢA. Then the pBAD/gⅢA- lvast was transformed into E. coli. After L-arabinose induction, the fusion protein was confirmed by SDS-PAGE and Western-blot analysis. Polyclonal antibodies were prepared using purified recombinant protein, which had a relatively high tite(1: 10000).(3) Analysis of the interaction among BP53, LVAST and VP37 by ELISA, competitive ELISA and Dynabeads. Results showed that no combination between LVAST and WSSV-VP37. BP53 could interact with astakine or WSSV-VP37 separately. While, VP37 inhibited the interaction between LVAST and BP53, which was dose dependent. A conclusion was drawn that WSSV-VP37 competed LVAST with BP53.(4) Analysis of recombinant LVAST's function in shrimp litopenaeus vannamei against WSSV by in vivo neutralization assay. Results showed that recombinant LVAST delayed WSSV infection for two days when compared with positive control groups, which indicated that providing recombinant LVAST delayed WSSV infection in shrimp.(5) Effect on viral replication of lvast by RNAi and Real-time PCR assay. RNAi technique was used to knock down endogenous lvast mRNA expression by sequence specific dsRNAs, then followed WSSV infection. vp37 was used as target gene to detect WSSV's replication in Real-time PCR assay, andβ-actin as the internal control. Results were analyzed using ??CT relative quantitative method. The amount of initially vp37 was took as 1, the vp37 mRNA expression level in positive control group reached the highest level on the fifth day post WSSV infection. In recombinant- LVAST-treated group, the vp37 mRNA expression level was eighty percent lower than that of positive control group in five days. And the vp37 mRNA expression in recombinant- LVAST-treated group reached the highest level on the sixth day post WSSV infection, in first five days, the vp37 mRNA expression were twenty percent of that of ositive control group. In lvast -dsRNA-treated group, the vp37 mRNA expression reached the highest level on the fifth day post WSSV infection, which were 2-fold higher than that of positive control group. In summary, recombinant astakine had some effect on inhibiting of WSSV's replication, and knocking down endogenous astakine gene could speed WSSV's replication.In summary, the results revealed that WSSV-VP37 competed astakine with BP53. The interaction between LVAST and BP53 was inhibited by WSSV-VP37, which was dose dependent. The functional studies of astakine in WSSV infection will help us better understand the life cycle of WSSV.