Cloning And Immunological Function Of Exopalaemon Carinicauda Hemocyanin Subunit With Higher Molecular Weight And Its Variants

The ridgetail white prawn Exopalaemon carinicauda is one of the important commercial shrimp species naturally distributed in the coasts of the Yellow Sea and the Bohai Sea,China.Due to the merits of extensive environmental adaptability,high reproductive ability,fast growth and wide feeding habits,the culture scale of E.carinicauda has expanded in recent years.E.carinicauda has showed obvious aquaculture prospects with the broad of the farming scale.However,the environmental deterioration,disease outbreaks caused by bacteria and virues had caused serious economic losses.The immune defense mechanism of shrimp and the disease resistance effectively are very important to aguaculture industry.E.carinicauda can only rely on the innate immune system such as cellular immunity and humoral immunity to prevent or clean out pathogen,as it does not possess acquired immunity.Hemocyanin is a multifunctional protein with various immunological activities in shrimp and other invertebrate's reported in recent years.Nevertheless,the sequence and immune function E.carinicauda hemocyanin subunit and its variants remain unclear.For above reason,the function of E.carinicauda hemocyanin subunit and variants was detected using the approaches of molecular biology and immunology.1.Cloning and expression analysis of hemocyanin subunit with higher molecular weight(EcHcL)The hemocyanin subunit cDNA of E.carinicauda(named EcHcL)cloned by the technology of rapid amplification of cDNA ends(RACE).The EcHcL cDNA was 2192 bp in length,including 2034 bp open reading frame(ORF)that encoded a 667 amino acids polypeptide of which contained a signal peptide sequence(1-21 amino acid residues).The predicted molecular weight of the mature peptide was 78.5kDa.The homology analysis showed that the amino acid sequence of EcHcL was highly conserved with other arthropod.The homology similarities of EcHcL amino acids with Macrobarchium nipponense hemocyanin and Litopenaeus vannamei amino acid sequence were 87% and 73%,respectively.The M domain of the amino acid sequence between EcHcL and other shrimp similar up to about 90%.The tissue expression analysis revealed that EcHcL detected in all tested tissues including gill,ovary,hepatopancreas,heart,intestine,muscle,stomach,abdominal ganglion,eyestalk and haemocytes,and the highest expression of EcHcL was in hepatopancreas.Quantitative Real-time PCR analysis indicated that the expression of EcHcL was upregulated distinctly in the hepatopancreas and haemocytes of shrimp after pathogen infection.After Staphylococcus aureus challenge,EcHcL transcripts level significantly increased in hemocytes and hepatopancreas at 48 h and 24 h,2 times than that in the control group.The maximum value occurred at 12 h and 24 h after Vibrio parahaemolyticus and white spot syndrome virus(WSSV)challenge,and its expression was 2-8 times as high as that of the control group.RNAi results showed that the expression of EcHcL significantly lower than the control group in hepatopancreas and haemocytes after injection of dsEcHcL and infection pathogens.The results suggested EcHcL showed different temporal expression patterns and might play an important role in shrimp immune system.2.Cloning and expression analysis of three EcHcL variantsThree EcHcL variants(EcHcL1-2-3)based on E.carinicauda transcriptome library were obtained using RACE technique.The total length of EcHcL1-2-3 were 2248 bp,2121bp and 2245 bp,respectively,which encoded protein of 685,676 and 676 amino acids,respectively.EcHcL1-2-3 are relatively conservative in evolution,and has typical domains of hemocyanin,including copper ion binding region,conserved His site and Ig-like region.EcHcL1 may related to pigment metabolism and phenoloxidase activity because of more than a tyrosinase domain.The results of tissue expression distribution showed that EcHcL1-2-3 were expressed in all of 10 tissues,and higher expressed in hepatopancreas and haemocytes.Different variants of E.carinicauda hemocyanin subunit may also have various immunological functions.The expression of the 3 variants of mRNA after pathogen stresses were investigated using the Real-time PCR strategy.The results showed that EcHcL1 presents higher expression level during 12-48 h after Staphylococcus aureus,Vibrio parahaemolyticus and WSSV stress,and its among more 1.5-4 times higher than that in matched group.EcHcL2 and EcHcL3 present higher expression level among 24-48 h after stress,which increased 2-9 times and 2.5-9 times compared with the control group.Therefore,we speculated that the E.carinicauda hemocyanin subunit variants may related to immune defenses closely and might have antimicrobial resistance activity.3.Prokaryotic expression and antibacterial effect analysis of EcHcL3Based on the above results,EcHcL3 was selected to conduct the prokaryotic expression and bacteriostasis experiment because it is significantly related to the immune correlation of pathogenic infection.The expression vector of pET28a-EcHcL3 was constructed and induced to obtain recombinant protein rEcHcL3 with 80 kDa that consistent with expected size.The results of mass spectrometry showed that it matched with the original sequence successfully.The results of bacteriostasis experimental showed that rEcHcL3 has extensive inhibitory effect on gram-positive bacteria and gram-negative bacteria.The rEcHcL3 can inhibit the 25.9%,24.8%,18.6%,41.5% and 21% growth of Staphylococcus aureus,Escherichia coli,Vibrio parahaemolyticus,Vibrio harveyi and Vibrio anguillarum respectively in the low of concentration of 0.01 mg/mL,which suggesting that it has strong inhibitory effect on bacteria.And the results provided important significance for understanding the immunological mechanism of multifunctional protein of hemocyanin.