Cloning And Expression Analysis Of Immune Related Genes HSP70、TNFAIP8、GBP1 And TLR2 From Hyriopsis Cumingii

Hyriopsis cumingii is one of the specific mussel which exploited for freshwater pearl production in China. It’s economic benefit is vulnerable affected by a variety of disease threats. Based on that the shellfish only have the innate immune system, the study of Hyriopsis cumingii immune genes has of great significance. In the present study, the cDNA sequence of Hyriopsis cumingii heat shock protein 70(HSP70),tumor necrosis factor(TNF)-alpha-induced protein 8(TNFAIP8), interferon-induced guanylate-binding proteins1(GBP1) and toll-like receptor2(TLR2) were cloned by rapid amplification of cDNA ends methods based on their long chain sequence were determined by High-throughput transcriptome sequencing of H. cumingii blood cells.Results showed that the full-length of HcHSP70 cDNA was 2298 bp long including a 1974 bp open reading frame(ORF) encoding a polypeptide of 657 amino acids, estimated molecular mass of 71.6 kDa and an isoelectric point of 5.67.Sequence comparison indicated that HcHSP70 shared the highest identity(91%) with HSP70 of Tegillarca granosa and Crassostrea gigas. HcHSP70 m RNA constitutively expressed in 5 detected tissues(hepatopancreas, gonad, gill, mantle, muscle), with the highest expression showed in hepatopancreas. By using qRT-PCR and Western blot,we detected both mRNA and protein of HcHSP70 in gill significantly up-regulated at37°C, and rapidly down to normal level under 40 ℃.The full-length of HcTNFAIP8 cDNA was 1179 bp including a 573 bp open reading frame(ORF) encoding a polypeptide of 190 amino acids, estimated molecular mass of 23.64 kDa. HcTNFAIP8 has highest similarity with CgTNFAIP8 was 71%,and them were on the same branch of the phylogenetic tree. The expression of HcTNFAIP8 in hepatopancreas down-regulated obviously injected LPS, than back to nomal range. But the expression of Hc TNFAIP8 has not affected after injected PGN.The full-length of HcGBP1 cDNA was 2418 bp including a 1947 bp open reading frame(ORF) encoding a polypeptide of 648 amino acids, estimated molecular mass of76.28 kDa. The full-length of Hc TLR2 cDNA was 1177 bp including a 1032 bp open reading frame(ORF) encoding a polypeptide of 343 amino acids, estimated molecular mass of 39.58 kDa.