The Study Of Cloning,Expression And Analysis Of Antibacterial In Carassius Auratus Of Histone H2B And HLP 1

The diseases control and prevention in aquaticulture has been the focused attention. Although have obvious antibacterial effects, antibiotics, once were used too long term, the problem of drug-resistant pathogens will appear. Therefore, some agents were in need to take place of the traditional antibiotics. As a kind of natural feed additives, antimicrobial peptides have ability to overcome the deficiencies of traditional antibiotics. In this study,we got the full-length H2 B gene from crucian carp in Qihe river by molecular cloning, then extracted a partial sequence from the N-terminal sequence of the coding region in H2 B which named HLP1, and analyzed its molecular characteristics. Besides that, the RT-qPCR technique was used to detect their expression in different tissues and response to A.hydrophila. At last, prokaryotic expression of HLP1 and the antibacterial activity of synthetic HLP1 were researched.In this study, the primers for middle segment were designed from conservative sequences of coding region in channel fish(letalurus punetaus). The total RNA was extracted from the kidney, gill, spleen,liver and other tissues from Carassius carp in Qihe river, then the first-strand cDNA was obtained by RT-PCR as the template to amplify the middle segment from the gene H2 B in carp; The target sequences was connected to pMD19-T vector and transferred into DH5α. At last, the 375 bp middle segment of histone H2 B was obtained successfully. According to the middle fragment, Two pairs of specific primers were designed for 3?UTR and 5?UTR; At last, 3?UTR(410bp) and 5?UTR(32bp) were amplified by nested PCR.Then the 817 bp full lenth sequences of histone H2 B from carp was obtained. It was found that the ORF of histone H2 B has 375 bp and encodes 124 amino acids.Using Online software to analyze histone H2 B,found that the protein was unstable with a molecular of 13608.8Da; The PI is 10.37 and has 18 units of positive charge. The secondary structure of histone H2 B contains a large of α-helix and random curl, someβ-folding and extension. The homology of the histone H2 B genes was analyzed using software BioEdit. We analyzed the coding region of histone H2 B from 14 species including zebra finch(Taeniopygia guttata)which had a distant relationship, then concluded that they had a homology more then 75 percentage, the homology was highest between zebrafish and Carassius carp in Qihe river with 90.7 percentage.The polypeptide HLP 1 comes from the N-terminal of histone H2 B, which has a large number of basic amino acids. First, to get HLP 1 sequence, we designed a pair of specific primers according to thefull-length sequence of histone H2 B, and successfully constructed the recombinant vector PET-32a-HLP 1.Then PET-32a-HLP 1 was transformed into E.coli BL21. Second, we provoked the positive colonies into LB medium containing AMP to culture, until the OD600 reached 0.6 to 0.8. After induction culture by IPTG,the cells were precipitated, washed and splited. Lysates were collected and the fusion protein was detected successfully by SDS-PAGE. Third, this experiment examined the expression of histone H2 B gene and HLP1 gene in gills, kidney, spleen, liver and head kidney by RT-qPCR, and we found that the mRNA content in liver, kidney and head was from high to low, with a relatively lower expression in gill and spleen. The expression of HLP1 was highest in head-kidney, higher in liver and same with H2 B in last tissues. After an Aeromonas hydrophila injection to fish, in 48 h, the expression of H2 B gene and HLP 1 gene in each tissue was significantly upregulated.We get the HLP1 via synthesis and diluting with a concentration gradient of 25μg/mL, 50μg/mL,100μg/mL and 200μg/mL. As the concentration of HLP 1 increasing, the diameter expanded gradually in antibacterial circle experiments. The 25μg/mL of HLP 1 may have an equal antibacterial activity with100mg/mL of AMP, which indicates a high antibacterial activity of HLP 1. It implied that HLP 1 might play an important role in immunity and antimicrobial of aquatic organisms, especially fish.