Cloning And Expression Of Hypodermin A Gene In BL21E.coli

Warble flies of the genus Hypoderma caused important economic losses are common parasites and very prevalent in many countries. During its migratory phase, the parasite release three serine protease: Hypodermins A, B, C.It has been demonstrated that these secreted larval enzymes are involved in the control of larva survival and in escape from the immune system. Hypodrmins are good candidates for immunotherapy and for use as serodiagnostic reagents .Hc is the most ideal antigen of them, while HA with better antigenic character also shows the greatest immunomodulatory activity on the host immune system. The incidence of warble infestation is declining in many countries, and it is becoming more difficult to find a reliable supply of larvae. Additionally, the use of antigens prepared from larva collected from the field in different countries leaves the possibility of batch-to-batch variation, for these reasons recombinant antigens look an attraction. In this paper ,the gene coding for the HA wais amplified from PGEM-HA by PCR using the oligonuceotide primers with Hindu and Not I restriction site at both ends for cloning purposes. The DNA fragment about 835bp purified from the Agrarose was ligated into the expression vector PET-28b,then,transformed into E.eoli TG I host bacteria. The positive recombinant was obtained through comparing the molecular mass of plasmids , identification of recombinant plasmids by PCR,digesting by restriction endonuclease HindIII,Not I and sequence analysis. The results showed that the recombinant (PET28b-HA) contained the HA gene with correct sequence. The identified positive recombinant was transformed into E.coli BL21 host bacteria, The IPTO-induced recombinant bacteria could express HA fusion protein with a predicted molecular weight of 31 KDa.peaks of target protein was achieved at 6h after adding 48 IPTG, the fusion protein purified by Ni-NTA metal chelate affinity presented onemajor band in the SDS-PAGE and reacted with rabbit serurn raised against the HA inwestern blotting, the fusion protein could not degrade gel8tin when the gelatin wasused as substrate.The results showed that the constructed PET28b-HA could express in E.coli BL2lhost bacteria, the HA fusion protein has reactiongenicity without enzyme activity, andthese provided the basis for further studying the different characters of HA protein.