Cloning, Expression And Characterization Of Serine Protease Inhibitor Gene Of Haemonchus Contortus

Sheep is an important livestock species, widely distributed throughout in China. The numbers of feeding volume, according to the statistic, is ranked first in the world. However, because of extensive management, the number of parasite incidence and prevalence of large, according to the survey, the goats and sheep suffering from win Haemonchus contortusis up to 50%-93.85%, to livestock caused enormous losses, seriously affected sheep industry. Haemonchus contortus belongs to Haemonchus genus of Trichostrongylidae family. It resides abomasums and small intestines of ruminants, causing anemia syndrome, and the sheep infected lead to anemia, weight loss and weakness, even death.Serine protease inhibitor is one of the natural component of the immune system, belonging to serine protease inhibitor superfamily protein, which involved in blood coagulation reactions, inhibiting protease activity, secretion of inflammatory cell, melanization, reproductive, apoptosis, tumor metastasis, signal transduction, and other important physiological processes cascades. Serine proteases together serine protease inhibitors, during parasite infection and innate immune responses of hosts, play an important role. Their reactions of coordination with each other in the signal transduction and cascade, regulat the biochemical reactions and metabolic process, such as blood coagulation, protein hydrolysis and signal transduction. Therefore, the cloning and characterization of the serine protease inhibitor gene of Haemonchus contortus, is benefited to investigate the mechanism of parasites invasion and against parasites immune response, development of anti-parasite immunology.In this present research, using rapid amplification of cDNA ends, serine protease inhibitor gene (hcserpin) of Haemonchus contortus is cloned at first time, and the sequence analysis suggeat that it is a novel gene belonging to serpin superfamily protein. After recombinant protein expressed with prokaryotic system, purified recombinant protein HCSERPIN is obtained utilization of affinity chromatography and size exclusion chromatography. Then the characteristics research of HCSERPIN is sequentially preformed as follows: 1. GENE CLON AND SEQUENCE ANALYSIS OF SERPIN FROM HAEMONCHUS CONTORTUSSerine proteinase inhibitor (Serpin) of H.Contortus belongs to the serine protease inhibitor family of proteins that are nearly found in all groups of organisms. The cascade reactions elicited by serpins are involved in a number of fundamental physiological and pathological processes in parasites and hosts. In order to clone the full length sequence of H. Contortus serine proteinase inhibitor (hc-serpin) cDNA, a new gene of H. contortus, specific primers for rapid amplification cDNA ends (RACE) were designed based on the expression sequence tag (EST, BM173953) to amplify the 3’-and 5’-ends of hc-serpin. The full length of the cDNA of hc-serpin gene was obtained by overlapping the sequences of 3’-and 5’-extremities and amplification by reverse transcription-PCR. The sequence was submitted to GenBank and confirmed to be an unknown or new mRNA (Accession number: FJ888352). Theresults showed that the cloned full-length cDNA comprised 1317 base pairs (bp) and contained a open reading frame (ORF) with 1104 base pairs which showed sequence similarity to several known serpin genes. The sequence analysis of hc-serpin gene showed that it can translated a polypeptide with 367 amino acid (aa) residuces. The ORF of the full length cDNA is located from 28 to 1131 bp, and a nematode-specific type 1 leader splicing (SL1) sequence,21 base pairs, is present at its 5’end, which extended from 5 to 26 bp. The present of SL1 indicates that hcserpin lies in an operon in genomic DNA because the SL1 is a symbol of downstream gene of operon. After an interval of G, SL1 is followed by the longest putative ORF containing several start codons ATG, but most of all ORFs terminate at the same one stop codon. Detailed analysis of Hcserpin cDNA sequence showed that there were 3 protein kinase C (PKC) phosphorylation sites,4 Casein kinaseⅡ(CKⅡ) phosphorylation sites,3 Myristyl acylation (N-myristoylation) sites,2 Tyrosine kinase phosphorylation sites,4 ASN-glycosylation-sites (glycoprotein’s sign),11 O-GlcNAc glycosylation sites in its predicted protein of hcSerpin. The theoretical pI and Mw of the protein were 8.5 and 41016.17 Dalton, respectively. Furthermore, a transmembrane helix is present at the amino acid residues from 18-35 of the putative protein and non-potential cleavage site of signal peptide was found in the polypeptide fragment. That indicated the hcserpin was unexcreted in natural conditions. There were two highly conserved domain near the C-terminal,97.5% aligned, reactive site loop 1 (RSL1) GTTA 319-322, reactive site loop 2 (RSL2) FEANHP 344-349, respectively. Amino acid residues in RSL, which belong to the serpin protein family, inquiry against the protein reference data- base of GenBank by domain aligned on line. The results suggested that the full length cDNA sequence cloned by RACE is a novel gene sequence and belongs to the serpin protein family.2. PROKARYOTIC EXPRESSION OF HCSERPIN AND PURIFICATION OF RECOMBINANT PROTEINSpecific primers for PCR amplification ORF of hcserpin were designed based on the complete hcserpin DNA sequence (accession number:FJ888352). The product of reverse transcription PCR was cloned into PMD18-T vector. Sequence analysis was detected by BamH I and EcoR I digestion and sequencing. The ORF fragments were, which purified with DNA affinity column kit, subcloned into pET32a expression vector. Then positive recombinant plasmid was transformed into E.coli. DE3 competent cells. Ten randomly selected clones with inserts of the expected size were identified by BamH I and EcoR I digestion. Positive clones containing the ORF fragment were picked out and sub-cultured in Luria Bertani media supplemented with 60μgml-1 ampicillin and incubated at 37℃until an OD600 of 0.6 was achieved. Expression of recombinant plasmid pET32a-hcserpin was induced with Isopropyl-(3-D-thiogalactopyranoside (IPTG) in E.coli. DE3.The recombinant HCSERPIN protein was purified by affinity chromatography and size-exclusion chroma- tography approaches. The rats were immunized by subcutaneously injection with recombinant HCSERPIN for arising multiclonal antibody. The natural serpin of H. contortus and antibody against the rHcserpin of goats sera infected by H. contortus were detected with Western blotting approach.The results shown that the size of ORF is 1104 bp coincident with predictive size. The pET32a-hcserpin plasmid was transmitted into E.coli.DE3. The base on further research for expression and characterisation of hcserpin is cheerful established by the successful recombinant pET32a-hcserpin plasmid. The puified rHCSERPIN, which gave an apparent single protein band, is migrated as a protein of 60.5 kDa on SDS-PAGE gel. Western blotting assay indicated that rHCSERPIN induced a significant antibody reaction in rats. The natural HCSERPIN protein of H. contortus was distinguished by rat anti-rHCSERPIN antisera, but isomers were not identified by the rat anti-rHCSERPIN antisera. The natural HCSERPIN from H. contortus appeared to have a larger molecular mass than that of the rHCSERPIN expressed in E. coli. Furthermore, the rHCSERPIN was identified by the serum of goats naturally infected with H. contortus, but no protein of anti-rHCSERPIN in negative control was identified by the serum of normal goats.3. TRYPSIN INHIBITION EFFECT AND OPTIMAL REACTIVE CONDITIONS OF rHCSERPINThe rHCSERPIN refolded was diluted with 50 mmol/L PBS, pH 7.6. Concentration of recombinant preptein was quantified using spectrophotometer. The rHCSERPIN 50μg and trypsin 50μg, dissolved in 0.4 ml 0.1 M PBS, were incubated in water bath at 37℃for 30 min. Bovine serum albumin 150μg dissolved in 100μl of the same PBS buffer was added to the above reaction and incubated for another 60 min at 37℃. The controls contained either an equivalent amount of rHCSERPIN, BSA or trypsin alone. The reactions were stopped by application of denaturant at 100℃for 10 min. The proteolytic efficiency of trypsin were analyzed by 12% denatured gel SDS-PAGE. BAEE was utilized as substrate to detect the inhibition efficiency of Trypsin, optimal pH and temperature of rHCSERPIN. The results suggest that the activity of trypsin in the BAEE assay declined significantlyin contrast to the BAEE control. The highest inhibitory rate of Trypsin activity was declined at 52.3%. The BSA observed in gel was not degradation completely in rHCSERPIN reactive group, contrast to control group no BSA remains found. rHCSERPIN is a thermally inert protein. It maintained its proteolytic activity even above 75℃. Meanwhile, the rHCSERPIN was insensitive to an extensive range of pH in vitro. The hydrolytic activity of trypsin was inhibited by rHCSERPIN from pH2.0 to 10.0. The optimum pH for activity of H. contortus rHCSERPIN was found to be 7.6 in the present study.4. ANTICOAGULATION ACTIVITY OF rHCSERPIN IN VITROThe anticoagulation activity of rHCSERPIN was determined in vitro. Three rabbits were selected to determine the coagulation time of rHCSERPIN using tube test. The data were expressed as mean±standard deviation (t-test). The recalcification time and antithrombin time were registered for t-test in comptuer, respectively. Differences between groups were analyzed by the Excel 2008, with a p-value<0.05 considered significant. The results shown that the rHCSERPIN was provided with anticoagulation effects significantly, and prolonged the recalcification time and antithrombin time markedly(p<0.05, p<0.01). Blood coagulation in the presence of 50 ug/ml rHCSERPIN took double the time taken by control samples. The anticoagulation effect of rHCSERPIN appeared to be dose dependent in this investigation. 5. DISTRIBUTION OF HCSERPIN IN H.CONTORTUSAdult male and female H. contortus worms were collected from goats and washed in 0.01 mol/L PBS (pH 7.4) and fixed overnight in modified Bouins fluid The parasites were washed, dehydrated, embedded in paraffin wax and cut into 5μm thick sections, respectively. The HCSERPIN protein detection was performed by the immunohistochemical reaction, and the sections were examined by light microscopy. The result suggested that the intense immunostaining was localized to the epithelial cells of the gastrointestinal tract in adult female and male worms, especially on the dorsal (DS) and ventral (VS) surfaces and intestinal epithelium. However, no expression of the HCSERPIN gene was observed in other tissues on the sections, such as scarfskin epithelia, testes (male worms), or ovaries and eggs (female worms). The expression mode of HCSERPIN is coincidence in adult female and male worms. Taking these observations together, the results may be beneficial for investigation the true functions of HCSERPIN in vivo to increase our understanding of the biology and pathogenic mechanisms of H. contortus.