Molecular Cloning And Functional Characterization Of Haemonchus Contortus HC58cDNA Gene

Haemonchus contortus is a highly pathogenic parasite affecting sheep, goats andcattle, causing major losses to the agricultural industry world wide. The parasitebelongs to the phylum (nematoda), order (strongylida), super family(trichostrongyloidea), family (trichostrongylidae), and genus (Haemonchus). Controlcosts of H. contortus and other nematode parasites are estimated to be overUS$2,000million annually. Haemonchosis control is so far carried out usinganthelmintics and grazing management however, excessive and uncontrolled use ofchemical drugs have resulted to emergence of anthelmintic resistant strains of theparasite, toxic residues in the human food chain and environmental pollution. Theenormity of this problem and the low priority given to the development of new classesof anthelmintics by pharmaceutical companies has generated the need to developalternative strategies for the control of H. contortus parasite. Vaccination seems to bethe ultimate, effective and sustainable strategy to controlling this parasite. Severalvaccines have been tested for protection against H. contortus including the cathepsinB like (CBL) cysteine proteases. HC58 is an expressed sequence tag of H. contortus parasite. Its published partialsequence indicated it might be a cathepsin B (CBL) cysteine protease. Its ubiquitousin situ localization however, was different with previously characterized cathepsin Bsof H. contortus.The thesis describes cloning and sequence analysis of the full length HC58cDNAgene, prokaryotic expression, and confirmation of its natural product and functionalcharacterization of the recombinant HC58 protein. The humoral and cellular immuneresponses of goats vaccinated with HC58cDNA vaccine are discussed. The studieswere undertaken sequentially as follows:1. Cloning and sequence analysis of Haemonchus contortus HC58cDNA geneFive gene specific primers were designed based on H. contortus expressedsequence tag (EST) HC58 of GenBank acc. no. AF305964. The 3'end (495bp) and5'end (430bp) cDNA products were generated by 3' and 5'-RACE (rapid amplificationof cDNA ends) procedure, respectively. Total RNA was extracted from a pooledsample of male and female adult worms and used as template for each amplificationreaction. Synthesized 3' and 5'ends were purified separately using the "TakaraAgarose gel DNA purification Kit, Ver-2.0", and cloned into pMD-18T cloning vector.E. coli DH_5a was transformed with recombinant pMD-18T/ 3' or 5'end products,respectively. The recombinant pMD-18T/3' or 5'end plasmids were identified withXbaâ… and Hindâ…¢restriction enzyme digestion and gel electrophoresis. The 3' and 5'end inserts were verified by sequencing and were identical to H. contortus EST HC58of GenBank acc. no. AF305964. The full length sequence of HC58cDNA gene was obtained by joining overlapsequences of 5' and 3' ends with SEQ EDIT computer programme. The 851bp longHC58cDNA gene had open reading frame of 654bp encoding 217 amino acid proteinwith an isoelectric point (pI) of 8.431 and predicted theoretical molecular mass of24,332.70 dalton (～24kDa). The protein could easily be aligned with members ofpapain AC-family cysteine proteases.2. Expression of HC58 cDNA in E. coli and confirmation of its natural product.HC58cDNA of H. contortus was cloned into pET-28a prokaryotic expressionvector and E. coli BL₂₁ was transformed with the recombinant pET-28a/HC58cDNA.Expression of HC58cDNA was induced with isopropyl-B-D-thiogalactopyranoside(IPTG) at a final concentration of 1mM. After 4 hr of incubation at 37℃, bacteriacells were harvested and expression was assayed by SDS-PAGE. The expressionproduct was about 29kDa. The recombinant protein, formed an insoluble inclusionbody, was dissolved in 8Mol/L urea and sequentially dialyzed in PBS pH 7.4 buffercontaining digressive concentrations of urea. Western blot analysis of native proteinsof H. contortus probed with rat anti-recombinant HC58 serum displayed a single bandof～32kDa instead of the calculated molecular mass of～24kDa. This suggested thatnative HC58 protein was glycosylated. The peripheral membrane protein extract (p2)of H. contortus reacted strongly with rat-anti-HC58 serum at～32kDa suggesting thenative product of HC58 had peripheral membrane protein localization and was alsoglycosylated. The recombinant HC58 protein reacted with serum from goatsartificially infected with H. contortus, suggesting that native HC58 protein was secreted during the course of parasite infection and induced antibody responses ingoat.3. Hemagglutination activity of recombinant HC58 proteinThe hemagglutination activity of recombinant HC58 protein was detectedutilizing erythrocytes derived from healthy human blood samples of type AB, A, B, Oand blood samples from goat, dog, rabbit, chicken, mice and buffalo. The resultsshowed that recombinant HC58 protein was blood group specific and exhibited higherhemagglutination activities to human blood type AB, A, B, O and erythrocytes fromdog, rabbit, mice and buffalo, and lower activities to chicken blood at concentrationsof 400μg/ml and 1.6mg/ml. No agglutination was detected to erythrocytes of goat andchicken at protein concentrations of 80μg/ml. Recombinant HC58 protein did notagglutinate erythrocytes from the natural host of H. contortus, the goat, despiteutilizing high recombinant protein concentrations of 1.6mg/ml. This suggested thatHC58 protein might play a role in the survival of H. contortus.4. Cysteine protease activity of recombinant HC58 proteinThe cysteine protease activity of recombinant HC58 protein over a broad rangeof pH was examined in vitro. The protein hydrolyzed synthetic peptide substrates ofZ-FR-AMC (N-benzylcarbonyl-Phenylalanyl-Arganine-AMC) and Z-RR-AMC(N-benzyloxycarbonyl-Arginyl-Arginine-AMC) and had characteristics for thecysteine protease family of proteins. In the acidic pH range, the recombinant HC58protein actively degraded hemoglobin (Hb), goat IgG and azocasein. By contrast, itdegraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. The recombinant HC58 proteindigested native hemoglobin in vitro consistent with its putative in vivo activity. Thesefindings suggested that HC58 protein may play roles during blood meal feeding of theparasite.5. Vaccination of goats against Haemonchus contortus with HC58 DNA vaccineDue to critical functions in worm physiology, H. contortus cysteine proteases arepotential candidates for the immunological control of haemonchosis. However, thereis limited information on the vaccination potential of these molecules in goat. In thischapter, HC58 DNA vaccine, against adult H. contortus was evaluated for protectionagainst homologous challenge infection in goat. Sixteen goats were allocated into fourexperimental groups of four animals each. HC58 DNA vaccine was administered 28and 14 days before challenge with 5, 000 dose of infective H. contortus L₃ (thirdlarval stage), respectively. The results indicated that HC58 DNA vaccine wastranscribed at local injection muscle sites and expressed in vivo seven and ten daysafter primary and booster vaccination, respectively. Following a single L₃ challenge,group (mean±SD) eggs per gram feces (epg), percent L₃ hatched from eggs andabomasum worm count for group 1 (HC58 DNA vaccinated goats) were reduced by28.02ï¼…, 47.6ï¼…and 28.3ï¼…, respectively. Corresponding reductions for group 2(recombinant HC58 protein vaccinated goats) were 26.36ï¼…, 31.6ï¼…and 16.1ï¼…respectively, (P＜0.05). Accordingly, there was a slight but significant difference inabomasum worm count for vaccinated goats in group 1 and 2 of 532.25±143.54 and622.5±71.29 respectively, compared to group 4 (unvaccinated positive controls) of 741.5±241.45, P＜0.05. Vaccinated goats mounted high serum IgA, IgG and mucosalIgA antibody responses subsequent to L₃ challenge. The findings suggested that HC58DNA vaccine has presented protective role in vaccination of goat against H. contortusparasite infection.6. Cellular immunity in goats vaccinated with HC58 DNA vaccine againstHaemonchus contortusVaccination with DNA vaccines against adult H. contortus induces significantlevel of protection to homologous infection in goat. To date however, mechanism ofprotection is not well understood, especially in goat. In this section, HC58 DNAvaccinated goats were artificially infected with 5, 000 dose of infective H. contortusL₃ (third larval stage), and cellular immune responses examined. The peripheral CD4⁺,CD8⁺T and B lymphocytes for vaccinated groups increased compared to unvaccinatednegative and positive controls after L₃ challenge infection. Likewise, mean eosinophiland lymphocyte levels increased substantially after vaccination and L₃ challenge. Onthe contrary, circulating neutrophil and white blood cells reduced under similarexperimental conditions in goats carrying an equal L₃ nematode burden. The resultssuggested that regulation of H. contortus expulsion in goat is a complex mechanismorchestrated by CD4⁺ and CD8⁺T cells, recruitment of eosinophil and lymphocytesand inclined towards development of Th₂ responses.