[The effect of Dermatophagoides pteronyssinus group 2 allergen T cell fusion epitope on STAT6 signaling in mice with asthma].

Objective: To investigate the alterations of signal transducer and activator of transcription factor 6(STAT6) signaling in a mouse model of asthma receiving treatment with Dermatophagoides pteronyssinus group 2 allergen(Der p 2) T cell fusion epitope and the mechanisms of the specific immunotherapy. Methods: Thirty mice were randomly divided into three groups by the random number table method: the asthma group, the treatment group receiving immunotherapy with Der p 2 T cell fusion epitope, and the negative control group (PBS group)(n = 10 in each group). Mice in the asthma and the treatment groups received intraperitoneal (i. p.) injection of 100 µl Der p 2 solution [PBS containing 100 µg/ml Der p 2 and 2% Al(OH)3] on days 0,7 and 14, respetively, while mice in the PBS group received same volume of PBS containing 2% Al(OH)3. From day 21, 30-min steam inhalation of 0.5 µg/ml Der p 2 was applied to the asthma and treatment groups (once daily for 7 successive days), and the PBS group inhaled same volume of PBS. From day 25 to day 27, the mice in the treatment group received i. p. injection of 200 µl of Der p 2 T cell fusion epitope (100 µg/ml) while the PBS and the asthma groups received the same volume of PBS. Mice were sacrificed at 24 h after the last inhalation, the bronchoalveolar lavage fluid (BALF) collected, and the total protein was extracted from the lung tissue. The levels of IFN-γ, IL-4 and IL-13 in BALF were determined by ELISA. The expression of STAT6 and phosphorylated STAT6 (p-STAT6) in the lung tissue was detected by Western blotting. Data were analyzed with the one-way variance analysis (ANOVA) method. Results: The level of IFN-γ in the treatment group[(234.40 ± 24.46) pg/ml] was significantly higher than that in the asthma group[(155.80 ± 20.53) pg/ml](P < 0.01). The levels of IL-4 and IL-13 in the treatment group [(30.00 ± 5.50) pg/ml and (174.50 ± 25.99) pg/ml, respectively] were both significantly lower than those in the asthma group[(53.28 ± 8.26) pg/ml and (308.10 ± 28.32) pg/ml, respectively](P < 0.01). Similarly, the levels of STAT6 and p-STAT6 in the treatment group(0.803 ± 0.221 and 0.966 ± 0.323, respectively) were both significantly lower than those in the asthma group (1.669 ± 0.296 and 1.735 ± 0.298, respectively)(P < 0.01). Conclusion: The Der p 2 T cell fusion epitope may function through inhibiting STAT6 to treat asthma in mice.

[Immunotherapeutic Effect of Dermatophagoides pteronyssinus Group 1 Allergen T Cell Epitope Peptide Against Allergic Asthma in Mice].

Objective: To study the specific immunotherapeutic effect of Dermatophagoides pteronyssinus group 1 major allergen T-cell fusion epitope peptide vaccine TAT-IhC-DPTCE against allergic asthma. Methods: One hundred and twenty SPF-grade BALB/c mice were randomized into PBS group (group A), asthma group （group B）, and immune treatment groups respectively receiving intraperitoneal (i.p.) injections of ProDer p 1 allergen (group C), DPTCE （group D）, TAT-DPTCE (group E) or TAT-IhC-DPTCE (group F) （n=20 in each group）. In detail, PBS （group A） or allergen extract derived from Dermatophagoides pteronyssinus （groups B-F, 10 µg） was intraperitoneally injected on days 0, 7 and 14, and was continued by aerosol inhalation from day 21 for 7 consecutive days （0.5 µg/ml, once/day, 30 min each time）. The mice in groups C-F received i.p. injections of 100 µg/ml ProDer p 1, DPTCE, TAT-DPTCE and TAT-IhC-DPTCE respectively 30 min prior to inhalation challenge on days 25-27 as a specific immunotherapy, while those in groups A and B received 200 µl PBS. Twenty-four hours after the last inhalation challenge, all the mice were sacrificed. The lung histopathological changes were examined by HE staining. The levels of IFN-γ, IL-13, IL-10 and TGF-ß in the bronchoalveolar lavage fluid (BALF) was determined with ELISA, and eosinophils in the BALF were counted (n=20 mice in each group). The serum level of IgE, IgG1 and IgG2a in orbital blood was determined by ELISA（n=5 mice in each group）. Results: HE staining revealed increased BALF eosinophils and decreased pulmonary inflammation in group F compared with group B. The IFN-γ level in group F ［（298.75±26.09） pg/mlï¼½ was significantly higher than those in groups B［（158.71±20.89） pg/mlï¼½, C［（210.38±18.92） pg/mlï¼½, D ［（229.44±13.00） pg/mlï¼½ and E［（233.24±20.39） pg/mlï¼½ （all P<0.01）. Similar results were also found for IL-10 and TGF-ß, while the IL-13 levels in groups C ［（47.35±4.71） pg/mlï¼½, D ［（41.90±4.28） pg/mlï¼½, E［（41.05±6.50） pg/mlï¼½ and F［（18.53±5.67） pg/mlï¼½ were all significantly lower than that in group B ［（66.68±6.63） pg/ml］（all P<0.01）. The number of BALF eosinophils in group B ï¼»5.65±0.91ï¼½×105/mlï¼½ was significantly higher than that in group A ［（0.45±0.39）×105/mlï¼½ （P<0.01）, while the BALF eosinophils in groups C ［（4.00±0.59）×105/mlï¼½, D ［（3.39±0.63）×105/mlï¼½, E ［（3.24±0.69）×105/mlï¼½ and F ［（1.42±0.49）×105/mlï¼½ decreased after immune treatment (all P<0.01). ELISA results showed that the serum IgE level in group F ［（5.26±1.72） ng/mlï¼½ was significantly lower than those in group B ［（32.81±2.98） ng/mlï¼½ and the other 3 treatment groups [group C, （20.06±3.17） ng/ml; D, （17.06±3.18） ng/ml; E, （16.23±3.61） ng/mlï¼½. Similar results were also obtained for IgG1. In contrast, the serum IgG2a level in group F［（43.10±1.34） ng/mlï¼½ was significantly higher than those in group B［（12.61±1.87） ng/mlï¼½ and the other 3 treatment groups ï¼»group C, （23.37±2.67） ng/ml; D, （25.60±2.10） ng/ml; E, （25.91±1.33） ng/mlï¼½ （all P<0.01）. Conclusion: Immunotherapy with chimeric TAT-IhC-DPTCE can effectively ameliorate the allergic airway response and pulmonary inflammation in mice.

[Exploration on the Technology of Counter Staining of Fluke].

Fixed samples of Clonorchis sinensis and Fasciolopsis buski were stained with acetocarmine and malachite green, or stained with acetocarmine only. The samples displayed three different colors after staining with acetocarmine and malachite green. The digestive system, excretory system and the surrounding muscle tissue were stained reddish, the uterus was bright green, and the vitellarium at each side of the worm was tan. Staining with the two dyes resulted in clear structure and moderate degree of staining, and allowed three-dimensional observation, while staining with acetocarmine highlighted the testis tissue. Therefore, combination of the two staining methods is recommended in teaching and research to more effectively facilitate observation.

[Cloning and Prokaryotic Expression of Fusion Gene of Group II Allergen Der p2 T Cell Epitope from Dermatophagoides pteronyssinus].

OBJECTIVE: To express and purify the T cell epitope fusion peptide of the major allergen Der p2 from Dermatophagoides pteronyssinus. METHODS: Nucleotide sequences reported to encode four T-cell epitopes (T1-T4) of Der p2 of D. pteronyssinus were linked in the rank of T1-T2-T3-T4. In this way, the chimeric gene was synthesized, named as Der p2 T. The gene of Der p2 T was amplified by PCR, purified, and cloned into the pET-28a (+) vector, forming the prokaryotic recombinant expression vector pET-28a (+)-Der p2 T. This formation was verified by double digestion. The pET-28a (+)-Der p2 T vector was transfected into E. coli strain BL-21, and its expression was induced by addition of IPTG. The recombinant protein was purified and collected by Ni-NTA affinity chromatography, and prepared for SDS-PAGE and Western blotting analysis. ELISA was used to evaluate the binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy. RESULTS: Double digestion results confirmed the construction of the pET-28a (+)-Der p2 T vector. SDS-PAGE revealed the expression of recombinant Der p2 T cell epitope fusion peptide with M, of 10,000. Western blotting confirmed the purification of Der p2 T cell epitope fusion peptide. The binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy [(37.70±9.89) µg/ml] decreased significantly in comparison to that of Der p2 [(85.89±9.63) µg/ml] (P<0.01). CONCLUSION: The Der p2 T cell epitope fusion peptide is prepared, and its binding ability to serum IgE from patients with house dust mite allergy significantly decreases than that of Der p2.

The complete mitochondrial genome of the brown leg mite, Aleuroglyphus ovatus (Acari: Sarcoptiformes): evaluation of largest non-coding region and unique tRNAs.

The circular mitochondrial genome (mitogenome) of Aleuroglyphus ovatus was sequenced. It was 14,328 bp long, and consisted of 37 coding genes including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. This is the first description of the complete mitogenome of a species in the Acaridae (Acari: Sarcoptiformes). The mtDNA gene order for A. ovatus is identical to those of Dermatophagoides farinae and D. pteronyssinus, but distinctly different from the mtDNA of other Acari. Most inferred tRNA genes of A. ovatus are extremely truncated (48-62 bp), lack stem-loops on either the T- or D-arm (except the trnK), and are unable to fold into the canonical tRNA cloverleaf structure. The largest non-coding region (378 bp) contained several conserved sequences involved in the regulation of mitogenome replication, including one core sequence (ACAT) associated with termination of the J-strand replication and several hypothetical stem-loop structures. The microsatellite-like (AT)n sequence in the largest non-coding region was observed in two other Astigmata species, but it has not been found in other Acari.

[Prevalence of metacercariae of Clonorchis sinensis in wild freshwater fishes from Nenjiang River around Qiqihaer City].

From May to November 2013, a total of 1175 wild freshwater fishes were collected from the rivers of Chuoer, Yalu, Wuyuer, Alun, and Yin in Nenjiang River basin Qiqihaer City, and examined for metacercariae by direct compression method. The metacercariae were collected by artificial digestion method. Forty Kunming mice were infected with 30-40 metacercariae of Clonorchis sinensis. The mice were sacrificed 36 days after infection, and the adult worms were collected from bile duct, and observed under microscope. The results showed that a total of 1 175 fishes, belonging to nine species were taken from the Nenjiang basin of Qiqihaer region. The infection rate of Clonorchis sinensis metacercariae was 51.2% (602/1 175). All the species were infected besides Silurus asotus, and the highest prevalence (82.7%, 91/149) was found in Longnose gudgeon and the lowest (7.1%, 6/84) in Perccottus glenii. Among the rivers, the highest prevalence of metacercariae was in Wuyuer River. (65.7%, 218/332), and the lowest was in Alun River and Yin River (24.1%, 67/278) (P<0.05). Each part of the body in the Carassius auratus and Pseudorasbora parva were susceptible for metacercariae. The main infection site in Longnose gudgeon was the fish scales, and C. sinensis metacercaria was first discovered in the brain tissue of Phoxinus lagowskii. The experimental results showed that the adult worms of C. sinensis were found in the hepatic bile duct of the mice, with an infection rate of 85.0% (34/40). The suckers, digestive system and reproductive system of C. sinensis were visible clearly.

[Seasonal changes of glycogen level in Oncomelania hupensis].

Oncomelania hupensis snails were collected from Qingyi River of Wuhu City from August 2012 to July 2013. Livers and pedal muscles of snails were dissected. Anthrone colorimetric method was used to evaluate the glycogen concentrations of whole-body, liver and muscle. The concentration of whole-body and liver glycogen decreased from September to next June. The whole body glycogen content in female (0.55 microg/mg) and male (0.88 microg/mg) snails was the lowest in June and April, respectively. The mean whole-body glycogen concentration in females and males was 2.99 and 3.39 microg/mg, respectively. Liver glycogen concentration was lowest in May (female = 0.29 microg/mg, male = 0.22 microg/mg), and reached peak level in August (female = 2.49 microg/mg, male = 2.78 microg/mg). The average liver glycogen concentration in female and male snails was 1.09 and 0.89 microg/mg, respectively. The muscle glycogen concentration gradually decreased from February to June, the lowest was found in June (female = 0.25 microg/mg, male = 0.41 microg/mg), and reached peak level in December (female =16.59 microg/mg, male = 10.06 microg/mg). The average muscle glycogen concentration in female and male snails was 799 and 605 microg/mg, respectively. There was a positive linear correlation between whole-body and liver glycogen concentrations (P < 0.05), and both of them had the similar trend in their monthly change. A positive linear correlation was found among whole-body, liver and muscle glycogen concentrations (P < 0.05).

[Experimental study on the Der f 1 mRNA molecules derived from dermatophagoides farinae for specific immunotherapy on murine model of asthma].

OBJECTIVE: To investigate the effect of Der f 1 mRNA molecules for specific immunotherapy on murine model of asthma. METHODS: Fifty BALB/c mice were randomly divided into 5 groups: PBS group, Der f 1 sensitization group, Der f 1 specific immunotherapy (SIT) group, beta-actin mRNA SIT group, and Derf 1 mRNA SIT group. On days 0, 7 and 14, mice in PBS group received PBS injection; mice in the other groups were intraperitoneally injected with 10 microg Derf 1. At day 21, the mice in the 4 experimental groups were challenged with a 30-min inhaled dose of Der f 1 (100 microg/ml) for 7 successive days. Two weeks after the final sensitization, the mice in the above five groups were im- munized by intradermal injection with PBS, 1 microg Der f 1, 10 microg Der f 1, 2 microg beta-actin mRNA, and 2 microg Der f 1 mRNA, respectively for 3 times at one-week intervals. Two weeks after the last intradermal injection, all mice were sacrificed and bronchoalveolar lavage fluid (BALF) was collected. ELISA was performed to detect the levels of IFN-gamma and IL-13 in BALF, the number of eosinophils in the BALF was recorded. Splenocytes were prepared, and cultured with Der f 1 al- lergen (10 Jg/ml) for 72 h. Splenocytes of PBS group was cultured without Derf 1 allergen. The levels of IFN-gamma and IL-13 in splenocyte culture supernatant were measured by ELISA, as well as serum antibody levels of total IgE, allergen- specific IgE (sIgE), sIgG1, and sIgG2a. Lung sections were stained in hematoxylin and eosin, and observed under the microsope. RESULTS: Except for PBS group, mice in the other 4 group showed symptoms of acute asthma attack. Com- pared with Derf 1 sensitization group [(897.56 +/- 105.73) pg/ml] and beta-actin mRNA SIT group [(219.47 +/- 64.72) pg/ml], the level of IFN-gamma in BALF from Der f 1 mRNA SIT group [(897.56 +/- 105.73) pg/ml] and Derfl SIT group [(864.48 +/- 70.62)pg/ml] significantly increased (P<0.01). However, the level of IL-13 in BALF from Derf 1 mRNA SIT group [(241.64 +/- 31.41) pg/ml] and Derf 1 SIT group [(321.94 +/- 41.07)pg/ml] was significantly lower than that of Der f 1 sensitization group [(520.62 +/- 43.77) pg/ml] and beta-actin mRNA SIT group [(507.22 +/- 42.26) pg/ml](P<0.01). The number of eosinophils in Der f 1 mRNA SIT group [(1.33 +/- 0.44) x 10(5)/ml] and Der f 1 SIT group [(1.48 +/- 0.39) x 10(5)/ml] was also lower than that of Der f 1 sensitization group [(3.54 +/- 0.52)x10(5)/ml] and beta-actin mRNA SIT group [(2.98-0.53) x 10(5)/ml] (P<0.01). The levels of IFN-GAMMA and IL-13 in splenocyte culture supernatant showed that IFN-gamma level in Der f 1 mRNA SIT group [(420.91+69.92) pg/ml] and Der f 1 SIT group [(334.92 +/- 43.72) pg/ml] was significantly higher than that of Der f 1 sensitization group[(123.75 +/- 5.48) pg/ml] and beta-actin mRNA SIT group[(128.84 +/- 59.00) pg/ml] (P<0.01). However, IL-13 level of Der f 1 mRNA SIT group [(268.51 +/- 40.42) pg/ml] and Der f 1 SIT group [(285.26 +/- 62.21) pg/ml] was significantly lower than that of Derf 1 sensitization group [(613.89 +/- 51.54) pg/ml] and beta-actin mRNA SIT group [(524.05 +/- 39.12) pg/ml] (P<0.01). Compared with Der f 1 sensitization group [total IgE: (94.34 +/- 11.66) ng/ml, sIgE: (65.67 +/- 9.47) ng/ml, sIgG1: (75.18 +/- 9.52) ng/ml, sIgG2a: (2.81 +/- 1.17) ng/ml] and beta-actin mRNA SIT group[total IgE: (86.48 +/- 10.26) ng/ml, sIgE: (62.36 +/- 8.35) ng/ml, sIgG1: (69.51 +/- 8.98) ng/ml, IgG2a: (1.06 +/- 0.11) ng/ml], the serum antibody levels of total IgE [(33.72 +/- 9.78) ng/ml], sIgE [(22.76 +/- 8.09) ng/ml], sIgG1 [(17.87 +/- 7.59) ng/ml] of Der f 1 mRNA SIT group decreased significantly (P<0.01), whereas the level of IgG% [(7.74 +/- 0.88) ng/ml] increased (P<0.01). Compared with Der f 1 sensitization group, the asthmatic symptoms were relieved after immunization with Der f 1 mRNA for specific immunotherapy, including intact structure of respiratory and alveolar epithelial cells, decreased inflammatory cell infiltration, and similar to those in Der f 1 SIT group. However, the breakage and detachment of bronchial epithelial cells occurred in beta-actin mRNA SIT group. CONCLUSION: Derf 1 mRNA vaccine can correct Th1 and Th2 imbalance.

[Effect of immunotherapy with recombinant allergen group 3 from dermatophagoides farinae in asthma mice].

OBJECTIVE: To explore the effect of specific immunotherapy with major 3 group recombinant allergen rDer f 3 of Dermatophagoides farinae in murine asthma model. METHODS: Forty BALB/c mice were randomly divided into 4 groups, namely PBS group (negative control), ovalbumin(0VA) group (positive control), rDerf3 allergen sensitization group (asthma group), and rDerf3 specific immunotherapy group(SIT group). The mice in asthma group and SIT group were injected intraperitoneally with purified rDer f 3 protein on days 0, 7 and 14, respectively, and rDer f 3 solution was inhalated from day 21 for 7 days. During the 25th-27th day, mice in SIT group were injected subcutaneously with 100 jg rDer f 3 allergen for 30 min before nasal inhalation. Mice in groups of PBS and OVA were treated with PBS and OVA, respectively. Twenty-four hours after the final challenge, all mice were sacrificed, the bronchoalveolar lavage fluid (BALF) was collected and the total number of white blood cells and the number of eosinophils were recorded. The levels of IL-5 and IFN-gamma in BALF and supernatant of cultured splenocytes were detected by ELISA, as well as the serum levels of specific IgE and IgG2, antibodies. Lung tissues were stained with haematoxylin and eosin for histological analysis. RESULTS: Compared with the asthma group, the rDer f 3-induced lung inflammation was significantly alleviated in SIT group. The total number of white blood cells [(7.03 +/- 1.38) x 10(8)/ml] in SIT group was considerably lower than that of OVA group [(22.11 +/- 3.70) x 10(8)/ml] and asthma group [(22.75 +/- 3.24) x 10(8)/ml] (P<0.01). The change trend of eosinophil leukocytes was similar with that of white blood cells. IL-5 levels in BALF [(108.20 +/- 11.02) pg/ml] and splenocyte culture supernatant [(98.34 +/- 13.06) pg/ml] in SIT group were significantly lower than that of OVA group [(182.04 +/- 13.94) pg/ml, (208.26 +/- 10.63) pg/ml] and asthma group [(195.33 +/- 15.33) pg/mL, (179.54 +/- 13.65) pg/ml] (P<0.01). Whereas, the level of IFN-gamma in BALF [(107.98 +/- 12.64) pg/ml] and supernatant of cultured splenocytes [(105.51 +/- 1.62) pg/ml] in SIT group was significantly higher than those of OVA group and asthma group (P<0.01). Compared with OVA group [(26.87 +/- 4.30) IU/ml] and asthma group [(35.25 +/- 8.84) IU/ml], a lower level of allergen-specific IgE [(9.12 +/- 3.78) IU/ml] and higher level of allergen-specific IgG2, [(38.52 +/- 6.33) microg/ml] were observed in SIT group (P<0.01). CONCLUSION: rDer f 3 allergen can reverse allergen-induced airway and lung inflammation in murine asthma model.

[Experimental study on polysaccharide of Cipangopaludina chinensis against HBV in vitro].

OBJECTIVE: To evaluate the biological activity of polysaccharide of Cipangopaludina chinensis (PCC) against HBV in vitro. METHOD: HepG2 2. 2. 15 cells were taken as the in vitro experimental model. The cell toxicity was observed by MTT. PCC of different safe concentrations and positive control medicine 3TC were added into the cells. Cell control without medicine was set at the same time. Cultural supernatants were collected at 9 d. HBsAg and HBeAg in cultural supernatants were tested by ELISA. The content of HBV-DNA was detected by TaqMan probe fluorescence quantitative PCR. RESULT: TC0 and TC50 of PCC in HepG2 2. 2. 15 cell culture were 1 g . L-1 and >10 g . L-1, respectively, suggesting low toxicity in cells. IC50 of PCC in HepG2 2. 2. 15 cells HBsAg and HBeAg were 0. 501, 0. 401 g. L-1, with SI being >19.96 and >24. 94, respectively. PCC could effectively inhibit the secretion of HBsAg and HBeAg, and have a better effect on HBeAg than on HBsAg. PCC had a significant inhibitory effect on HBV-DNA in HepG2 2. 2. 15 cells at concentrations of 0. 1, 1 g . L-1 P <0.05). CONCLUSION: PCC has the effect against HBV activity in vitro to some extent, with low toxicity, thereby having a good prospect for application.

[Investigation on species and community ecology of cheyletoid mites breeding in the stored traditional Chinese medicinal materials].

OBJECTIVE: To investigate the species of Cheyletoid mites breeding status in the stored traditional Chinese medicinal materials and the relationship between its community and habitats. METHODS: A total of 60 samples of the traditional Chinese medicinal materials were collected from Huainan, Wuhu and Xuancheng in China. The mites were isolated with shakesieve shock, directicopy, Tullgren funnel, waternacopy and redricopy, and identified and counted under the light microscope. RESULTS: Cheyletoid mites were represented in 48 of the 60 samples, and the breeding rate accounted for as high as 80.0% (48/60). Totally, 6 species of Cheyletoid mites were identified, which belonged to Cheyletus, Acaropsis, Cheletomorpha, Eucheyletic and Pseudocheyles genera under the family of Cheyletidae. In the three investigated areas, the average breeding density of Cheyletoid mites was 109.75 heads/g, and average richness index was 1.54. The index of species diversity was 2.55 and the number of evenness was 0.95. CONCLUSION: This result entails positive prevention and control of the mite breeding in storage and processing of herbal materials.

[Applying multiple displacement amplification to DNA typing in the pathological section].

OBJECTIVE: To investigate the feasibility of applying multiple displacement amplification (MDA) to DNA typing in forensic pathological section. METHODS: Ninety-eight pieces of pathological sections were prepared in terms of 3 factors as the period of preservation, tissue types and death ages, and randomized into groups by Latin square by double 7-order design. Silicon bead method was used to extract the DNA template. Compared with the PCR amplification performed directly by AmpFlSTR Identifiler kit in the control group, MDA was performed before amplification in the experimental group. Based on the samples from fresh autopsies as the standard genotypes, the number of detection and the detection rate were analyzed and compared between the experimental group and the control group. RESULTS: Between the control group and the experimental group, there was significantly statistical difference regarding the rate of DNA typing in each period of the tissue sections preserved (P<0.01). The detection rate of the 16 loci in the experimental group was more than 95% when the period of the tissue sections were preserved within 360d. There was significant difference in different tissue types (P<0.01). But there was no significant difference in different death ages (P>0.01). CONCLUSION: MDA is efficacious in DNA typing of forensic pathological sections, for it can improve the DNA template quantification through abating the inhibiting factor's concentration of PCR and reducing the rate of allele drop out (ADO). However, the period of the sections preserved and tissue types would affect the results of genotyping by MDA.

[Prokaryotic expression of chimeric gene derived from the group 1 allergens of dust mites and bioactivity identification].

OBJECTIVE: To express a chimeric gene R8 derived from the group 1 allergens of dust mites using prokaryotic expression system and detect their bioactivities. METHODS: PCR amplification was performed using specific primers of Derf1 gene and the pUCm-T recombinant plasmid containing the R8 chimeric gene as a template. The PCR products were inserted into the pET28a(+) empty vector after double digestion using restriction endonuclease BamH I and Xho I, respectively. The recombinant plasmid was transferred into E. coli line BL21 and induced by 1 mmol/L isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed product was detected by SDS-PAGE and the target protein was purified. IgE binding assay of the purified protein R8 was detected by ELISA using dust mite allergic patient sera. For determining immunogenicity of R8 protein, 75 BALB/c mice were randomly divided into 5 groups, namely PBS (negative control), rDer f 1 group and rDer p 1 group (positive groups), R8 group and asthma group. The mice were treated with dust mite extract at 0, 7, 14 day by intraperitoneal injection of allergens (100 jl, 0.1 .tg/jl) and inhaled challenge as aerosol (0.5 microg/ml, 30 min/d) on day 21 for 7 days. Before inhalation in immunotherapy groups at 25-27 day, specific allergen immunotherapy was performed using rDerf 1, rDerp 1 and R8 allergens respectively. Mice in negative control group were treated with PBS all the time. Twenty-four hours after the last challenge, mice in every group were sacrificed. The bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the level of interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) in BALF. RESULTS: SDS-PAGE analysis revealed that chimeric gene R8 was expressed with a band of approximately M(r) 35 000. Compared with groups of rDerf 1 and rDer p 1 [(80.44 +/- 15.50) and (90.79 +/- 10.38) microg/ml, respectively], IgE binding capacity of the protein R8 (37.03 +/- 12.46) microg/ml was statistically lower (P < 0.001). The level of IFN-gamma 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 < or = 46.11)pg/ml, respectively] (P < 0.01), but no statistical difference to that of the rDerf 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). CONCLUSION: Chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.

[Sequence analysis on drug-resistant gene of rpoB in MDR-TB among pneumoconiosis patients complicated with tuberculosis in Huainan mining district].

OBJECTIVE: To study the characteristics of drug-resistant genetic mutation of rpoB in multiple drugs resistant bacillus tuberculosis (MDR-TB) among patients of pneumoconiosis complicated with pulmonary tuberculosis. METHODS: A total of 114 clinical isolated strains of Mycobacterium tuberculosis were collected, MDR-TB were identified by conventional antimicrobial susceptibility test (AST). Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. RESULTS: The results by AST showed that there were 31 strains of MDR-TB in the 114 clinical isolated strains, the rate of drug resistance was 27.19% (31/114). No mutation of rpoB was identified in 10 rifampicin-sensitive strains that randomly chosen, while conformation changes were found in MDR-TB strains, and the mutation rate of rpoB was 93.55% (29/31) in resistant strains, mainly concentrated in codon 531 (45.16%, 14/31) and 526 (29.03%, 9/31), happened base substitutions, including 27 unit point mutation and 2 two point mutation. CONCLUSIONS: The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for RFP resistance in MDR-TB among patients of pneumoconiosis complicated with pulmonary tuberculosis. It also proves that rpoB gene is diversiform.

[Extraction of agglutinin from Oncomelania hupensis and its haemagglutination activity].

OBJECTIVE: To explore the extraction methods of agglutinin from Oncomelania hupensis snail and study its haemagglutination activity. METHODS: Protein obtained by ammonium sulfate fractionation precipitation with 20%-100% saturation of ammonium sulfate. Its haemagglutination activity was determined by rabbit erythrocytes. The precipitation which could agglutinate rabbit erythrocytes was diluted with 2.5 mg/ml D-galactose, D-fructose, D-glucose, saccharose, maltose and lactose, respectively, and then their haemagglutination activity was tested. Snail agglutinin were incubated at different temperatures (25-90 degrees C) and assayed for agglutinating activity. The effect of pH on the haemagglutination activity was determined by using the PBS buffer at different pH values (3.0-10.0). RESULTS: Oncomelania snail agglutinin exhibited high haemagglutination activity in 20%-40% saturated ammonium sulfate pellet. Lactose and galactose could inhibit the haemagglutination activity of snail agglutinin. The agglutinin showed maximum activity at pH 7.0. In temperature range of 30-70 degrees C, the haemagglutination activity decreased with increasing temperature, and all activity lost beyond 80 degrees C. CONCLUSION: Galactose/lactose specific agglutinin exists in Oncomelania snail, its haemagglutination activity is affected by pH and temperature.

[Reform and practice on the experiment teaching of medical parasitology].

A new model of education is investigated to meet the new idea of experiment teaching in university. Therefore the establishment of experiment teaching model of medical parasitology needs to be correspondently reformed. A variety of new management measures are taken to raise the efficiency of experiment teaching in training the students in the College.

[Isolation and purification of Oncomelania hupensis agglutinin and determination of its molecular weight].

OBJECTIVE: To isolate and purify agglutinin from Oncomelania hupensis snail and determine its molecular weight. METHODS: Agglutinin was preliminarily isolated from snail tissue homogenate by 0%-40% saturated ammonium sulfate, and then successively purified with Sephadex G-75 gel filtration and Sepharose 4B affinity chromatography. Bradford assay was used to determine the protein content. The agglutination activity was determined by rabbit erythrocytes. The purity of agglutinin preparations was assessed by SDS-PAGE. The molecular weight of agglutinin subunit was determined by Sephadex G-75 gel filtration. RESULTS: The specific activity of snail tissue homogenate was 21.74 titer/mg. After ammonium sulfate precipitation, Sephadex G-75 gel filtration and Sepharose 4B affinity chromatography, the specific activity of snail agglutinin from the homogenate solution increased to 61.93 titer/mg, 75.89 titer/mg and 963.86 titer/mg, respectively. SDS-PAGE analysis indicated that snail agglutinin (M, 53,000) was purified by Sephadex G-75 gel filtration and Sepharose 4B chromatography. The molecular weight of the snail agglutinin produced by Sephadex G-75 gel filtration was Mr 78,000. CONCLUSION: Combined use of salt fractionation, gel filtration and affinity chromatography can be efficient for extraction and purification of agglutinin from Oncomelania hupensis species. The snail agglutinin is characterized as mono subunit protein with a molecular weight of Mr 78,000.

[Gray sequence analysis of ecological factors in relation to Tyrophagus putrescentiae].

OBJECTIVE: To make gray sequence analysis of the ecological factors to the population fluctuation of Tyrophagus putrescentiae. METHODS: Samples of T. putrescentioe and its natural enemy were collected, examined and counted in every ten days, relative humidity and temperature were recorded simultaneously. Data were analyzed by gray sequence analysis. RESULTS: The population of T. putrescentiae in the wheat warehouse reached peaks in late June and mid-September respectively, and that of the predatory mites showed a following efficiency. The gray sequence of the ecological factors to the larvae of T. putrescentiae was temperature>natural enemy>relative humidity; to the nymphs and adults, it was relative humidity>natural enemy>temperature; and to the population, it was relative humidity>temperature>natural enemy. CONCLUSION: Relative humidity, temperature and natural enemy were important ecological factors affecting the population dynamic of T. putrescentiae, and the prevalence curve of T. putrescentiae shows a bimodal pattern.

[Anti-mite activity and skin safety of Herbal taraxaci extract for Demodex folliculorum].

OBJECTIVE: To test the in vitro effect of the extract of Herbal taraxaci on Demodex folliculorum. METHODS: Active Demodex folliculorum were obtained from patients with moderate to severe demodex infestation. Herbal taraxaci and Radix stemona were extracted respectively with 80% ethanol under 85 degrees C, and a preparation with a concentration of 200mg/ml was made. The extractions were used in vitro to examine the anti-mite activity by observing time of killing mites. Physiological saline and Radix stemona extraction served as blank control and positive control respectively. PH value of Herbal Taraxaci extract was noted. Skin irritation test of normal and wounded skin and acute toxicity test were carried out with rabbit shin, and Herbal taraxaci and 75% ethanol were served as experiment and control medicine. RESULTS: Motion and morphology of the mites considerably changed with the effect of Herbal taraxaci extract. The time of mite-killing was (1.50+/-0.65)min with Herbal taraxaci and (3.53+/-1.04)min with Radix stemona respectively (P<0.01) and over 120 min with physiological saline. pH value of Herbal taraxaci extract was 5.00+/-0.28. Score for irritation to normal and wounded rabbit skin was 0 and 0.3 respectively, and acute toxicity test showed no significant toxicity. CONCLUSION: Herbal taraxaci extract shows a remarkable in vitro activity to Demodex folliculorum with skin safety.