Transgenic Expression Characterization Of Aminopeptidase H11 From Haemonchus Contortus Using Caenorhabditis Elegans

The gastrointestinal nematode Haemonchus contortus is a major blood feeding pathogen of sheep and goats, which inhabits the mucosal surface of the abomasum in these ruminants and causes haemonchosis. Its infections may be manifested as anemia, weight loss or even death in some cases. Current control of Haemonchosis mainly depends on the use of anthelmintic drugs. But the increasing problem of resistance to all classes of anthelmintics highlights the need for novel control strategy, such as vaccination. No vaceine has yet been commercialized, due to the complicated life-cycle and incomprehensible components of antigens. Aminopeptidase H11 is an integral membrane glycoprotein which is present only in the intestinal microvilli of L4 and adults worms, and considered to be one of the best characterized hidden antigens from H. contortus (on average a greater than 90% reduction in faecal egg counts and a greater than 75% reduction in worm burdens). But no effective protection was provided by vaccination with the recombinant forms expressed in E. coli or baculovirus. The free-living nematode Caenorhabditis elegans has been validated as an alternative expression system to attempt to express proteins similar to their native forms, as it is closely related to H. contortus in evolutionary terms. Here, the study was aimed to characterizing genomic structure of Hll gene from H. contoruts, and to establish a transgenic platform for functional exploitation of genes and expression of antigen candidates from parasitic nematodes, taking C. elegans as a heterologous system, then to estimate the transcriptional activity of H11 5' flanking region and make sure the feasibility in expression of Hll using C. elegans, which could provide a novel strategy for vaccination against parasitic namatodes.1. Characterization of genomic structure of Hll gene from H. contorutsReferred to the sequence of Hll from H. contortus reported by Smith (1997), RT-PCR was carried out to amplify the cDNA of Hll, taking total RNA of H. contortus as a template. Alignment with its similarities from other organisms revealed a typical characterization of microsomal aminopeptidase with a zinc-binding motif HEXXHXW. Specific primers were designed, according to the conserved domains among these similarities, to amplify the genomic DNA of H11, employing the long-distance PCR and genome walking technique. The results revealed the open reading frame of H11 was 2919 bp in size, while the genomic DNA was 14,959 bp in size which contained 25 exons separated by 24 introns, and canonical GT-AG consensus sequences were at each intron-exon splice junction. These findings provided interesting materials to our further research.2. Establishment of a transgenic platform to exploitation of gene function and vaccine development of parasitic nematodes, taking C. elegans as a heterologous systemTo establish the transgenic platform, the core promoter regions of Act-1 gene and T07F10 gene, and the Poly (A) signal region of T07F10 gene were amplified from the genomic DNA of C. elegans using PCR, and SV40 early mRNA poly(A) signal sequence and EGFP gene were amplified from eukaryotic vector pEGFP-N1, respectively. Taking the vector pBluescript SK+ or pEGFP-4.1 as backbone, several varied recombinants were constructed, three of which, that is recombinant Pact-EGFP, pB-Pact-EGFP-T07F10T and pB-Pact-EGFP-SV40T, contained the core promoter region of Act-1 gene, and the others, named as recombinant pB-PT07F10-EGFP-T07F10T and pB-PT07F10-EGFP-SV40T, contained the core promoter region of T07F10 gene. After microinjection into the gonad of C. elegans together with pRF4 as a gene marker, only the core promoter region of Act-1 gene in vector Pact-EGFP could direct the expression of EGFP and green fluorescence was detected in the cortex, vice cortex and the pharyngeal of C. elegans. In contrast, the core promoter region of Pact-EGFP and pB-Pact-EGFP-SV40T could direct the expression of EGFP in Vero cell with different intensity, when transfected into Vero cell by liposome, which indicated that unique regulatory elements to transcription may exist in the 5'upstream of the core promoter or the 3'UTR of the gene. These results prompted greatly the further research on gene function of parasitic nematodes using C. elegans.3. Characterization of transcriptional activity of Hll 5'-flanking region, taking advantage of C. elegansTo proceed characterizing the transcriptional activity of Hll 5'-flanking region, genome walking was employed again to obtain more information of Hll 5'upstream. Alignment revealed the 1517-bp 5'-flanking region of Hll, and the last exon and its 3' UTR of an isoform H11-4. The result indicated that H11-4 had a tandem link to Hll gene, with the same orientation in gene extension in the chromosome. The 1517-bp 5'-flanking region of Hll was cloned upstream of GFP gene in ppd95.77 vector only or fused with the first two exons, the first intron and part of the second intron. After microinjected recombinant plasmid with 5'-flanking region of H11 into the gonads of C. elegans, the transformed animals exhibited fluorescence in the most anterior intestine and distal intestine in the L4 larvae and adult worms, which demonstrated different transcriptional patterns compared with that in H. contortus. The correct splice did not occur in C. elegans, when the first two exons, the first intron and part of the second intron of Hll gene was clone behind 5'-flanking region of H11, which was probably correlated with its unique function to the parasitism of H. contortus.4. Screening of promoters used to direct expression of heterologous gene and transgenic expression of Hll gene regulated by cpr-1 5'-flanking region in C. elegansThe 5'-flanking regions of three genes from C. elegans, that is T07F10.1a gene which codes the similarity of H11, cpr-1 and elt-2 gene which are only expressed in intestine, were screened to direct the expression of H11 in C. elegans. The 1885-bp, 1985-bp,1998 bp 5'-flanking region of these three genes were amplified and cloned upstream of GFP gene in pPD95.77 vector. After microinjection into the gonad of C. elegans, the progenies of trangenic worms were picked out and investigated. The results showed that the fluorescence directed by T07F10.1a 5'-flanking region is distinctively expressed in the intestine, excretory cells, nervous system and tail neurons in C. elegans of all stages but embryonic development, while directed by cpr-1 and elt-2 5'-flanking region were only detected in intestine, particularly in the L4 and adult C. elegans. cpr-1 5'-flanking region can direct intensive expression of GFP in the whole intestine. elt-2 5' flanking region direct the expression of GFP in the most-anterior intestine (int 1), mid-intestine and the distal intestine, and various differences in fluorescence intensity were shown among different individuals. cpr-1 5'-flanking region was determined to direct the expression of H11 gene, referred to tissue location of GFP, expression intensity in L4 and adult C. elegans and stability among different individuals.To obtain expression vector cpr-pPD95.77(G-), a specific pair of primers were designed. Taking advantage of PrimeStarTM SH DNA polymerase, GFP gene in recombinant vector cpr-1 5'-flanking region::GFP was removed employing long-distance PCR. The vector cpr-pPD95.77(G-) was formulated after self-ligation and cyclization of the amplification product with addition of four restriction enzymes. EGFP gene was subcloned downstream of cpr-1 5'-flanking region to verify the validation in expression of heterologous genes. Then, H11 gene and its partial sequence were cloned downstream of cpr-1 5'-flanking region into cpr-pPD95.77(G-), respectively, with 6 fold His tag added at both sides of the genes. SDS-PAGE and Western-blot were introduced to analyze the expression of both genes. Green fluorescence could be detected in the intestine at a high level, which indicated that recombinant vector cpr-pPD95.77(G-) also had the ability to express heterologous genes. The results suggested that it was not H11 but Trans-HPS could expressed in C. elegans, at the direction of cpr-1 5'-flanking region and specific bands termed Trans-HPS could be detected using both anti-His monoclonal antibody and polyclonal antibody, although Trans-HPS could not be purified using Nickel agarose gel affinity chromatography.5. Assessment of the immuno-protection of Trans-HPS against H. contortus in goatsLiquid culture was employed to bulk preparation of crude extraction from transgenic worms with Trans-HPS.250μg per goat of crude extraction was administered at primary immunization and booster two weeks later with the same dose. Assessment of the immuno-protection proceeded after orally challenged with 5000 infective H. contortus L3 2 weeks after the second immunization. High titre level of serum IgG was mounted just two-week post-primary immunization. Serum IgG levels reached peak values at two-week post-second immunization and remained significant titre until the end of this trial. Significant elevation in T cell proliferative responses stimulated by Trans-HPS was observed in both Trans-HPS immunized group and HPS immunized group. Faecal egg counts was reduced by 41.4% and the advent of the peak in FEC was markedly delayed in Trans-HPS immunized group. And worm burdens were reduced by 17.79%, which showed no statistical significance as positive control.In summary, the genomic DNA of H11 was first sequenced and characterized, and the tandem relationship between Hll gene and its isoform H11-4 was first revealed in our study; transgenic platform for exploitation of gene function and vaccine development of parasitic nematodes was developed and expression characterization and immuno-protection assessment of Trans-H11 proceeded, taking advantage of C. elegans. This system will be particularly important for expression of complex glycan epitopes and examination of their influence on the immune response to parasitic nematodes.