Expression Of CyPA Gene And Preparation Of Multiclonal Antibody In Hyriopsis Schlegelii

Hyriopsis schlegelii, originated in the Lake Biwa of Japan, was introduced to China in 1997. Compared with other freshwater pearl mussel, the H. schlegelii is advantage in rate of growth, disease-resistance and breed-pearling.Cyclophilins with a variety of functions have been widely identified in diverse organisms from microorganisms to mammals. It has two important functions, one is the cis-trans peptidyl-prolyl isomerase (PPIase), which catalytic protein folding and severs as molecular chaperones during protein transport processes. The other can suppress immunization in T cell. CyPA was the first number of Cyclophilins identified in 1984. Recently researchs indicated that the CyPA gene may play an immune role in infectied or stress conditions in some species. The full-length CyPA cDNA of Hyriopsis schlegelii (referred as HsCyPA) was identified from cDNA library of haemocytes challenged by Aeromonas hydrophila.To investigate the possible function of HsCyPA in freshwater mussel, Real-time PCR was employed to examine the mRNA expression in diffenent tissues and the haemocytes of different ages. The results showed that HsCyPA mRNA is ubiquitously expressed in all the tissue including haemocytes, heart, gill, foot, intestine, liver, kidney, gonad.The highest ones were liver and gonad, and the lowest ones were intestine and haemocytes.The results from the haemocytes of different ages showed that mRNA expression lever of HsCyPA is higher in the younger (one-and two-aged ones) than older (three-, four-and five-aged ones). Maybe protein folding and severs of younger is more than the older.For further research the function of HsCyPA in the haemocytes, liver and gonad tissues, the mRNA expression levers of this gene was examined by Real-time PCR at the time points of 0,2,4,8,16,24 and 48 h after A. hydrophila challenged in those tissues. The mRNA expression level of HsCyPA in haemocytes was almost stable during the process of challenge. While in gonad and liver, the expression of HsCyPA mRNA was significantly up-regulated after bacterial challenge at 4h, then droped. It suggested that CyPA from Hyriopsis schlegelii was not only a constitutive protein, but also an inducible protein, and perhaps involved in defending the pathogen infection.The complete ORF of HsCyPA subcloned into PET-32(a+) prokaryotic expression vector, and expressed in the coli prokaryotic expression system. The solubled fusion protein was purified by the poper inducement conditions and Ni-NTA affinity chromatography. The chymotrypsin-coupled assay showed that the fusion protein has the PPlase activity. The ELISA assay showed the potency of the polyclonal antibodies was 1:640000. The polyclonal antibodies were purified from the rabbits which were immunized by the fusion protein as antigen. All the results can provide some experiment technologies for further researchs about the functions of the HsCyPA gene.