CDNA Cloning And Prokaryotic Expression Of Paramyosin In Chorioptes Panda

Chorioptes spp.is one of the ectoparasites which is widely distributed in world.It parasitize on body surface,of many domestic animals and wildlives.The C.panda is one of the ectoparasites isolated from Panda.It is much hazardous for panda.For a long time,Methods available for the control of Chorioptes spp.infection are mainly based on chemotherapy which has adverse reaction and can produce many problems(such as chemical resistance and environmental pollution).Thus,study on new technique is needed.Base on the published nucleotide sequence of C.tezweus paramyosin gene and C. ovis paramyosin gene,a pair of PCR primers was designed and synthesized.Total genome DNA isolated from the C.panda strain was used as template to PCR amplification.The PCR production was cloned into PMD18-T vector,and 2628bp DNA fragment was obtained by sequencing.By blasting the homologous sequences in Genbank databases,DNA sequence was confirmed as the C.tezweus paramyosin gene sequence.The determined nucleotide sequences were compared pair wise for similarity,the results showed that the sequence of paramyosin in C.panda was 98.4% and 95.9%percent identify to the C.tezweus paramyosin gene and C.ovis paramyosin gene,was 90.8%percent identify to D.pteronyssinus paramyosin gene, and between 84.3%-81.3%percent identify to the S.scabiei paramyosin genes.This DNA sequence encodes a protein consisting of 875 amino acids(AA).The molecular mass of polypeptide was 102.581 kDa,its isoelectric point was 5.57,also has the strong hydroilicity and antigenicity.The sequence produced in this study was deposited in Genbank(Accession No.EU543652).By the technology of DNA recombination,the paramyosin gene was cloned into expression plasrnid pET32a(+),and was transformed to E.coli BL21(DE3).The recombinant bacteria were induced to expression through the changes of IPTG The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 122kDa,which was consisted of a 102kDa protein of paramyosin gene and pET-32a(+)(20kDa).The amount ofproteinum was to peak at 7h after IPTG induced. There were almost no difference to amount of proteinum in different IPTG concentration,all be high.Host immune responses to the recombinant proteins were determined by Western-blot analysis for individual animals using methods described previously on nitrocellulose strips with transferred paramyosin was expressed as the highest dilution showing immune reactive bands.