Expression And Function Study Of The Crustacean WAP Domain-containing Proteins And Bax Inhibitor 1-like Protein

As an important Crustacean, Chinese white shrimp, Fenneropenaeus chinensis and red swamp crayfish, Procambarus clarkii are good experiment materials which can be used to research the innate immune system of invertebrate. By researching the interrelated genes in the innate immune system, we can deeply know the immune principle in the antibacterial and antiviral responses in crustacean, which can provide new strategies for the disease control in the shrimp aquaculture. These researches can also provide data for understanding the innate immune system of invertebrate. In this paper, we obtained some immune related genes by random cDNA library sequencing. We chose the double WAP domain-containing protein gene of Chinese white shrimp, F. chinensis and the single WAP domain-containing protein gene and Bax inhibitor 1-like protein gene of red swamp crayfish, P. clarkii for further analysis and obtained following results. 1. Expression and functional study of the double WAP domain-containing protein gene in Chinese white shrimp, F. chinensis.The complete sequence of Fc-DWD was cloned from F. chinensis hemocytes. It had 787 bp in length with an open reading frame of 554 bp. The ORF encoded a protein of 117 amino acids. The mature Fc-DWD (101 amino acids) had a predicted molecular mass of 12.78 kDa and an estimated pI of 8.49. The whole molecule was composed mainly of double WAP domains in the carboxy-terminal of Fc-DWD. And there were 8 conservative cysteines in every WAP domain. In the transcription level, Fc-DWD transcripts were expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine in unchallenged shrimp. When challenged with bacteria and virus, the expression level of Fc-DWD obviously increased in hemocytes, hepatopancreas, gills, and intestine. Meanwhile, we recombinantly expressed the protein using the E. coli expression system and made the rabbit antiserum using the purified protein. According to the results of the binding assay to microorganisms, we found that rFc-DWD could bind to both Gram-negative bacteria (E. coli, P. aeruginosa, and V. anguillarum) and Gram-positive bacteria (S. aureus, B. megaterium, and B. subtilis). According to the results of the proteinase inhibitory assay, we found that rFc-DWD could inhibit the proteinase(s) activities produced by B. subtilis (Gram-positive bacteria) and P. aeruginosa (Gram-negative bacteria). So, according to the results of the expression profiles and the two vitro functions, we could speculate that Fc-DWD exerted important function in the innate immune system of the Chinese white shrimp, F. chinensis. And Fc-DWD might be an important immune effector molecule in the antibacterial and antiviral responses.2. Expression and functional study of the single WAP domain-containing protein gene in red swamp crayfish, Procambarus clarkii.The complete sequence of Pc-SWD was cloned and the full length was 998 bp. The ORF of Pc-SWD encoded a protein of 74 amino acids, with a 20 amino acid signal peptide in the N-terminus and a WAP domain in the C-terminus. And there were 8 conservative cysteines in the WAP domain. The mature Pc-SWD (54 amino acids) possessed a predicted molecular mass of 5.97 kDa and an estimated pI of 7.71. In the transcription level, Pc-SWD transcripts were expressed in the heart and intestine at a relatively high expression level and in hemocytes, hepatopancreas, and gills at a relatively low level. And Pc-SWD transcripts were not checked in stomach. In the translation level, Pc-SWD protein was highly expressed in the heart, hepatopancreas, gills, and intestine; it was relatively insignificantly expressed in the hemocytes of unchallenged P. clarkia. And the level of Pc-SWD protein in hemolymph was low. The expression profile of the Pc-SWD was researched in the mRNA level using the RT-PCR method after WSSV challenge. After WSSV challenge, the expression of the Pc-SWD transcript visibly increased in hemocytes, hepatopancreas, gills, and intestine. Meanwhile, the expression profile of the Pc-SWD was researched in the protein level using the Western Blot method after WSSV challenge. The results showed that Pc-SWD obviously increased in the hemolymph after WSSV challenge. The expression level of Pc-SWD obviously increased in the hemocytes and no obvious variation was observed in the heart, gills and intestine of WSSV-challenged crayfish. According to the results of the binding assay to microorganisms and the proteinase inhibitory assay, we found that the rPc-SWD which recombinantly expressed by using E. coli expression system in vitro, could strongly bind to both Gram-negative bacteria and Gram-positive bacteria. And the rPc-SWD could inhibit the proteinase(s) activities produced by B. subtilis and P. aeruginosa. So we speculated that the Pc-SWD might be an immunity interrelated gene in the innate immune system of the P. clarkii. And it might exert important function in the antiviral and antibacterial responses.3. Molecular cloning and expression profile of the Bax inhibitor 1-like protein gene in red swamp crayfish, Procambarus clarkii.The complete sequence of Pc-BI-1 was cloned and the full length of the cDNA was 1970 bp. The length of the open reading fragment (ORF) of Pc-BI-1 was 708 bp. And the ORF encoded a protein of 235 amino acids. There was no the signal peptide sequence in the N-terminus. And there was only a domain in the C-terminus, which was composed of 211 amino acids. The mature Pc-BI-1 (235 amino acids) possessed a predicted molecular mass of 25.9 kDa and an estimated p1 of 9.37. In the transcription level, the results of the qRT-PCR showed that the Pc-BI-1 transcripts were mainly expressed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine of the normal crayfish. And Pc-BI-1 transcripts were expressed in the hemocytes and heart at a relatively high expression level and in hepatopancreas, gills, stomach, and intestine at a relatively low level. We used the qRT-PCR method to study the Pc-BI-1 expression profile after bacteria and virus challenge, respectively. The results showed that the expression level of Pc-BI-1 obviously increased in hemocytes, hepatopancreas, gills, and intestine after bacteria and virus challenge, respectively. According to these variations of the expression profile, we speculated that Pc-BI-1 might be an immunity interrelated gene in the innate immune system of the P. clarkii. Pc-BI-1 might exert important function in the innate immune system and involve in the antibacterial and antiviral responses.