Yuxin Jiang

Production of a chimeric allergen derived from the major allergen group 1 of house dust mite species in Nicotiana benthamiana

Plants are widely accepted as a general platform for the large-scale production of recombinant proteins, which has been demonstrated by the successful expression of various exogenous proteins. Using plants as a bioreactor for mass production of target proteins for vaccines is thought to show the most potential. This study explores whether a chimeric allergen R8, derived from the major allergen group 1 of house dust mites species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), is expressed in tobacco. The highly efficient and useful Tobacco mosaic virus RNA-based overexpression (TRBO) vector was used to investigate expression of the R8 molecule in tobacco by agroinfection. Presence of R8 was detected using SDS-PAGE and Western blotting. Purified allergens were characterized using IgE-binding activity assay and allergen-specific immunotherapy (ASIT) in murine asthmatic models. The recombinant R8 was successfully expressed in tobacco leaves. The pro-peptide was observed in the herbaceous leaf extracts. This protein exhibits properties similar to the parental allergen ProDer f 1 expressed in Escherichia coli or tobacco with respect to IgE immunoreactivity. R8 also rectifies imbalance of TH1/TH2 cells. An herbaceous plant expression system model allows mass production of R8, which might be used in the future for diagnosis of asthma or production of a candidate vaccine for allergen-specific immunotherapy of asthma.

IFNG+874A/T polymorphisms and IFNG CA repeat polymorphism associated with asthma in Asian--a meta-analysis.

Abstract Introduction: Asthma is an etiologically complex disease and develops by the interaction of multiple genes and environmental factors. Previous studies suggested a linkage between the interferon-gamma (INFG) gene cluster in the chromosome 12q region and susceptibility to asthma. Methods: This meta-analysis was undertaken to examine the relationship between +874A/T and CA-repeat polymorphisms in INFG gene and asthma. Eligible articles were accumulated via online databases in October 2013. A total of nine articles were included that were associated with the +874A/T polymorphism, involving 470 cases and 574 controls, as well as articles involving CA-repeat polymorphisms, which included 365 cases and 517 controls. The meta-analysis was performed for heterogeneity and calculation of pooled odds ratio and evaluation of publication bias by Egger’s linear regression test. Results: The differences between IFNG polymorphism and the susceptibility to asthma may vary across ethnicities and ages, and also that the allelic frequencies of the AA genotypes in IFNG at +874 is associated with an increased risk of asthma in the Chinese Han adult population; additionally, CA14 occurrence in the first intron can increase the risk of asthma in the Japanese population. Conclusion: Our findings suggested that some polymorphisms in the IFNG gene could act as high prevalence-susceptibility markers of asthma. Larger scale studies are warranted to address these associations among different ethnic populations, ages and the severity of asthma prognosis.

Prokaryotic expression and bioactivity evaluation of the chimeric gene derived from the group 1 allergens of dust mites

BACKGROUND we successfully reconstituted the gene from group 1 allergens of dust mites, and obtained a body of shuffled genes. In order to verify the prediction on the chimeric gene, we tentatively cloned R8 into the vector that was prokaryoticly expressed, purified and assessed for its bio-activities. METHODS the expressed product was detected by SDSPAGE and the target protein was purified. The purified protein R8 was detected by ELISA. 75 BALB/c mice were divided into 5 groups, namely PBS, rDer f1, rDer p1, R8 and asthma group. The mice were treated with dust mite allergens at 0, 7, 14 day by intraperitoneal injection and inhaled challenge as aerosol on day 21 for 7 days. Specific allergen immunotherapy was performed using rDer f1, rDer p1 and R8 allergens respectively. The level of IFN and IL- 4 in BALF was detected by ELISA. RESULTS the chimeric gene R8 was expressed with a band of approximately Mr 35000. Compared with groups of rDer f 1 and rDer p 1 [(80.44±15.50) and (90.79±10.38) μg/ml respectively], IgE binding capacity of the protein R8 (37.03±12.46) μg/ml was statistically lower (P<0.001). The level of IFN in sera of R8 group [(343.43±38.79) pg/ml] was higher than that of the PBS and asthma groups [(393.93±50.68) and (208.44±46.11) pg/ml respectively] (P<0.01), but no statistical difference to that of the rDer f 1 and rDer p 1 groups (P>0.05). IL-4 level in R8 group was lower markedly than the others (P<0.05 or P<0.01). CONCLUSIONS chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.

CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.

UNLABELLED Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. METHODS the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. RESULTS the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. CONCLUSION we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Morphologic features of Sancassania berlesei (Acari: Astigmata: Acaridae), a common mite of stored products in China.

OBJECTIVE Sancassania berlesei (S. berlesei) is the leading threatening mite in breeding industry of Eupolyphaga sinensis and public health. METHODS Living specimens of S. berlesei were obtained from the surface of Eupolyphaga sinensis and purified with double-distilled water. The egg, larva, nymph, hypopus, adult male and female of S. berlesei were screened and picked out under microscope. RESULTS Morphological variations of S. berlesei, including its legs, setae, external genitalia and accessories, are clearly identified under SEM. The larva has three pairs of legs, with no leaf-like setae, yet its coxal rod is well-developed. By nymph stage, four pairs of legs and the fourth dorsal seta arise, whereas the genital area looks still under-developed. At hypopus, the claws and tarsules appear well-built, and leaf-like setae, setae of tibia and setae of genu are seen. The sucker plate totally contains nine suckers and four shell-like dimplings in which there are symmetric distributions with 1 pair of central suckers, 2 pairs of side suckers and 1 pair of anterior suckers, respectively. One pear-like posterior sucker is located at the back of sucker plate. All suckers are smooth except for anterior sucker with radial stripe. The genital sense organ of adults exhibits itself with cordiform external aspect and typical ossification texture; whereas the male is dissimilar with the female regarding seta number on the genital sense organ. CONCLUSION Description of the morphological structure in great detail for S. berlesei tends to supply the important information for the taxonomy and further study.