Molecular Cloning Of Thymosin β4 In Penaeus Monodon And Its Role In Antibacterial Immunity And Wound Healing

Thymosin β4,as a newly discovered important gene that can simultaneously inhibit bacterial growth and promote wound healing,has not been studied in P.monodon.In this study,thymosin β4 gene of P.monodon(Pmβ4)was cloned and its role in antibacterial immunity and wound healing was explored.The antibacterial activity of Pmβ4 in vitro was studied by liquid medium method and enzyme-linked immunosorbent assay.By RNAi and overexpression techniques,the clearance effect of Pmβ4 on V.parahaemolyticus in hemolymph and the effect of Pmβ4 on the survival rate were clarified.The relationship between Pmβ4 and key genes of wound healing(SOD and CAT)was explored through RNAi and overexpression.The effect of Pmβ4 on wound healing was further confirmed by tissue sections.The mechanism of Pmβ4 in innate immunity and wound healing of P.monodon was preliminarily revealed,which provided theoretical support for the infection and prevention of pathogens in the process of shrimp farming,and the repair of wounds.(1)The full length of Pmβ4 c DNA sequence was obtained in the transcriptome.The ORF is 501 bp and encodes 166 amino acids.The predicted protein molecular weight is 18714.05 D and the isoelectric point is 5.75.The results of multiple sequence alignment and phylogenetic tree showed that Pmβ4 of P.monodon was similar to thymosin β4 of other prawns and it had the closed evolutionary relationship with Penaeus japonicas.Tissue distribution revealed that Pmβ4 is abundantly expressed in immune organs such as hemolymph,lymphatic and hepatopancreas of P.monodon.(2)In this study,the antibacterial ability of Pmβ4 was studied in vivo and in vitro.In vitro,in the enzyme-linked immunosorbent assay(ELISA)experiment,when the protein concentration reached from 64μg / m L to 128μg / m L,the binding activity of V.parahaemolyticus,V.vulnificus,V.harveyi and Vibrio anguillarum showed high binding activity.The liquid culture method has strong antibacterial activity against Vibrio vulnificus,V.parahaemolyticus,Streptococcus agalactiae and S.aureus.The results showed that the recombinant protein Pmβ4 had broad-spectrum antibacterial activity and different sensitivity in vitro.In vivo,first of all,in the immunostimulation experiment,the expression levels of Pmβ4 showed an upward trend,reached a peak and remained relatively high after the injection of Vibrio harveyi,Vibrio parahaemolyticus and Staphylococcus aureus.In situ hybridization and q RT-PCR experiments proved that the ds RNA successfully interfered with Pmβ4.After injection of V.paralyticus,the hemolymph-coated plate method was used to estimate that the bacterial concentration of the interference group was significantly higher than that of the blank control group.At this time,the number of dead P.monodon was increased.The results of the overexpression group of recombinant protein Pmβ4 were contrary to those of the interference group,indicating that Pmβ4 participated in the immune defense process.(3)In order to study whether Pmβ4 has the effect of wound healing and explore how to promote the specific mechanism of wound repair.Firstly,the relative expression of Pmβ4 in hemolymph was analyzed after making the wound.It was found that Pmβ4 peaked after 12 h and has an obvious upward trend.It was preliminarily speculated that Pmβ4 is related to the recovery process of wound healing.Then the expression of wound healing genes was detected after 24 hours of silence and overexpression of Pmβ4.After wounding,it was found that the expression levels of CAT and SOD genes were significantly reduced after interference and significantly increased after overexpression.It is suggested that Pmβ4 may participate in the wound repair process by regulating the expression of antioxidant genes.(4)In order to deeply explore how Pmβ4 participates in the wound repair process,the wound condition of muscle was observed by tissue section.The damaged muscle tissue changed and was infiltrated by blood cells,surrounded by melanin in a circle and then the outermost layer began to necrosis.In the interference group,necrosis began to occur earlier than the blank group.However,no necrotic area was observed in the overexpression group.It shows that Pmβ4 can delay traumatic necrosis.It is fully proved that Pmβ4 may promote wound repair by regulating the transcription of wound healing genes and delaying necrosis.