目的原核表达尘螨主要变应原Der p 2。方法分离屋尘螨总RNA,根据GenBank已公布的Der p 2核酸序列设计引物用RT-PCR扩增Der p 2编码基因,克隆至pMD19-T载体、亚克... more 目的原核表达尘螨主要变应原Der p 2。方法分离屋尘螨总RNA,根据GenBank已公布的Der p 2核酸序列设计引物用RT-PCR扩增Der p 2编码基因,克隆至pMD19-T载体、亚克隆至表达载体pET28a(+),将表达质粒转化至Escherichia coli BL21(DE3)并用IPTG诱导表达,并对表达产物进行免疫印迹鉴定。结果成功构建了表达质粒pET28a(+)-Derp2,SDS-PAGE和Western blotting显示原核表达获得成功。序列分析表明所获得的Der p 2编码基因与参考序列同源性达99.54%,推测其编码氨基酸130个,相对分子质量约为14348.5。结论尘螨变应原Der p 2原核表达获得成功,为进一步生产重组变应原奠定了基础。
目的:获得尘螨变应原Derf2基因及其生物信息学资料.方法:提取粉尘螨总RNA,RT-PCR合成Derf2的cDNA片段,回收PCR产物并连接至pMD19-Tsimple,经测序验证后亚克隆入表... more 目的:获得尘螨变应原Derf2基因及其生物信息学资料.方法:提取粉尘螨总RNA,RT-PCR合成Derf2的cDNA片段,回收PCR产物并连接至pMD19-Tsimple,经测序验证后亚克隆入表达载体pET-28a(+),转化大肠杆菌感受态细胞后提取质粒,双酶切鉴定.用ExPaSy,EBI,NCBI网站的在线软件对测序结果进行分析.结果:RT-PCR扩增获得了Derf2cDNA片段,酶切、电泳结果表明克隆和亚克隆获得成功.与参考序列相比,测序结果多了87bp(77～163),但Blastn发现中国广州和德国Reinbek报道的序列中也含此87bp.同源性、相似性、序列比对及分子进化分析提示,该序列与中国广州和德国Reinbek报道的序列亲缘关系较近.推测该变应原由176个氨基酸组成,与附睾分泌蛋白E1具有同源性,信号肽位于1～17aa处,在6～24aa处有一跨膜螺旋.二级结构由a-螺旋(16.57%),延伸链(32.57%)和随机卷曲(50.86%)组成.结论:获得了粉尘螨变应原Derf2编码基因及其分子特征,为进一步生产基因工程变应原用于临床诊治变态反应性疾病奠定了基础.
目的:探讨哮喘患者血清免疫学指标及患者室内尘螨孳生密度的季节变化规律.方法:筛选螨性哮喘患者36例,于2005-09,12,2006-03,06采集患者卧室尘样计数尘螨,同步检测患者外周血总Ig... more 目的:探讨哮喘患者血清免疫学指标及患者室内尘螨孳生密度的季节变化规律.方法:筛选螨性哮喘患者36例,于2005-09,12,2006-03,06采集患者卧室尘样计数尘螨,同步检测患者外周血总IgE,s-IgE,s-IgG1,s-IgG2和s-IgG4.结果:哮喘患者血清总IgE,s-IgE,s-IgG1,s-IgG2和s-IgG4均高于正常对照组,差异均具统计学意义(P<0.01);比较患者血清总IgE,s-IgE及s-IgG1,s-IgG2,s-IgG4的4次检测值,差异均具统计学意义(P<0.01).患者血清总IgE,s-IgE在12月份检测值最高,6月份检测值最低;s-IgG1以6月份最高,3月份最低;s-IgG4在3月份最高,6月份最低.患者卧室尘螨孳生密度4次检测值差异具有统计学意义(P<0.01),以2006-03和2005-12为高.结论:哮喘患者血清总IgE,s-IgE,s-IgG1,s-IgG2和s-IgG4和卧室尘螨孳生密度呈季节变化,IgG1的变化趋势与尘螨孳生密度的变化相反,IgG4的变化趋势与尘螨孳生密度的变化一致.
目的克隆和分析屋尘螨主要变应原Der p 1,并实现原核表达。方法分离屋尘螨总RNA,根据Gen Bank已公布的Der p 1核酸序列设计引物,用RT-PCR扩增Der p 1编码基因,克隆至... more 目的克隆和分析屋尘螨主要变应原Der p 1,并实现原核表达。方法分离屋尘螨总RNA,根据Gen Bank已公布的Der p 1核酸序列设计引物,用RT-PCR扩增Der p 1编码基因,克隆至pMD19-T载体、亚克隆至表达载体pET-28a(+),将表达质粒转化至E.coliBL21(DE3)并用IPTG诱导表达,并对表达产物进行免疫印迹鉴定。结果成功构建了表达质粒pET-28a(+)-Der f 1,Western blotting显示原核表达获得成功。序列分析表明所获得的Der p 1编码基因与参考序列同源性达99.9%,推测其编码氨基酸222个。屋尘螨和粉尘螨1类变应原氨基酸序列相似率为60%,而粉尘螨与梅氏嗜霉螨1类变应原相似率为85%,分子进化分析亦提示粉尘螨和梅氏嗜霉螨亲缘关系较近。推测所获rDer p 1二级结构中,α螺旋占33.78%,延伸链占21.62%,随机线圈占44.59%。结论尘螨变应原Der p 1原核表达获得成功,为进一步生产重组变应原奠定了基础。序列分析表明粉尘螨和梅氏嗜霉螨的亲缘关系可能更近,而与屋尘螨关系稍远,此与现行的形态学分类系统并不符合。
该文综述了居室环境螨类的孳生率、分布、季节消长、种类,所致变态反应性疾病即螨性哮喘、过敏性鼻炎、异位性皮炎和慢性荨麻疹等的诊断和治疗,及其寄生于人体呼吸系统、消化系统、泌尿生殖系统及外耳道等处所... more 该文综述了居室环境螨类的孳生率、分布、季节消长、种类,所致变态反应性疾病即螨性哮喘、过敏性鼻炎、异位性皮炎和慢性荨麻疹等的诊断和治疗,及其寄生于人体呼吸系统、消化系统、泌尿生殖系统及外耳道等处所致的疾病,并简述了居室环境螨类孳生的控制措施及研究进展。
Objective: To characterize the group-29 allergens from
Dermatophagoides (D.) pteronyssinus and i... more Objective: To characterize the group-29 allergens from Dermatophagoides (D.) pteronyssinus and investigate their ability to cross-react with other group-29 allergens from D. pteronyssinus as well as those from D. farinae and Tyrophagus putrescentiae. Methods: Der p 29, Der f 29, and Tyr p 29 cDNA sequences were amplified from total RNA isolated from D. pteronyssinus, D. farinae and Tyrophagus putrescentiae, respectively. Then they were cloned into the pET28a vector, expressed in Rosetta2(DE3)plysS, and purified using anion exchange chromatography. The IgE-binding rates of rDer p 29 were assessed by IgE Western blotting. The four epitopes of rDer p 29 were predicted, synthesized, and detected by IgE-ELISA. The cross-reactivity among the recombinant proteins rDer p 29, rDer f 29, and rTyr p 29 was investigated using dot blot and IgE-ELISA inhibition experiments. The allergens’ physiochemical properties, amino acid sequences, and tertiary structures were also compared. Results: Der p 29 was successfully expressed in Rosetta2(DE3)plysS as a single, 393-bp open reading frame. Western blotting showed that the purified rDer p 29 protein exhibited an IgE-binding rate of 100% when tested on patient sera. The following four Der p 29 epitopes were predicted and synthesized: 37-45 (EP1), 57-69 (EP2), 75-80 (EP3), and 104-117 (EP4). IgE-ELISA tests on 20 D. pteronyssinuspositive sera yielded IgE-binding rates of 85% (rDer p 29), 80% (EP1), 55% (EP2), 40% (EP3), and 55% (EP4), respectively. The dot blot experiments further confirmed cross-reactivity among the three group-29 proteins. When used as an inhibitor, rDer p 29 demonstrated an average cross-reactive inhibition rate of 49.7% against rDer f 29 and 54.4% against rTyr p 29. When rTyr p 29 was used as an inhibitor, it showed an average cross-reactive inhibition rate of 56.3% against rDer f 29. Conclusions: A recombinant protein, rDer p 29 with strong allergenicity was produced. Moreover, it was found that rDer p 29 cross-reacted with rDer f 29 and rTyr p 29, due to their highly homologous sequences and structures. These findings highlight the importance of considering inter-species epitope cross-reactivity when diagnosing and treating allergic diseases.
The clustered regularly interspaced short palindromic repeats (CRISPR) structural family form an ... more The clustered regularly interspaced short palindromic repeats (CRISPR) structural family form an acquired immune system in bacteria that is able to degrade invading virus or phage DNA. The CRISPR/Cas system has been co-opted as an RNA-mediated nuclease for use in targeted genome engineering. The CRISPR/Cas system provides a novel tool for manipulating genomic DNA using an approach that is simple, convenient, and easy to learn. Since its first description as a genome editor in 2012, it has been widely applied to bacteria, yeast, nematodes, flies, zebrafish, mice, rats, and human cells. This review describes the basic principles of the system and its application. In particular, we focus on CRISPR as a tool to manipulate and study the functional genomes of the planet’s most abundant organisms, insects, which have diverse impacts on humans and the environment.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas, 2008
Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der ... more Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ss turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.
Aquaporins are a large family of transmembrane channel proteins that facilitate the passive but h... more Aquaporins are a large family of transmembrane channel proteins that facilitate the passive but highly selective transport of water and other small solutes across biological membranes. House dust mite () is the major source of household immunogens, and we have recently reported six cDNA sequence encoding aquaporins from this mite species. To better understand the structure and role of mite aquaporin, we constructed a tertiary structure for DerfAQP1 by homology modeling from the X-ray structure of malaria aquaporin PfAQP (Protein Data Bank code No. 3C02) and conducted molecular dynamics simulation. The simulation arranged seven water molecules in a single file through the pores of the DerfAQP1. Further, two conserved Asn-Pro-Ala motifs were located on Asn203 and Asn77; residues Arg206, Trp57, Met190, Gly200, and Asp207 constituted an extracellular vestibule of the pore; and residues His75, Val80, Ile65, and Ile182 constituted the cytoplasmic portions. The overall free energy profile for water transport through DerfAQP1 revealed an energy barrier of ~2.5 kcal/mol. These results contribute to the understanding of mite physiology and pathology.
Aquaporins are highly conserved water channel proteins involved in the transport of solutes acros... more Aquaporins are highly conserved water channel proteins involved in the transport of solutes across membranes. The few aquaporins identified to date play major roles in excretion in insects. Excretion is a major source of indoor allergens from the house dust mite species, but, to date, aquaporins have not been well studied in these species. We used RNA sequencing to study the transcriptome of the house dust mite Dermatophagoides pteronyssinus to identify putative aquaporin family members. RNA was isolated from more than 2000 mites for 5΄-RACE and 3΄-RACE. Putative aquaporins were identified through BLAST alignments, and primers were designed to amplify the five identified sequences. These cDNA fragments were then inserted into bacterial plasmids for molecular cloning and sequencing of DerpAQP1, DerpAQP2, DerpAQP3, DerpAQP4, and DerpAQP5 (GenBank accessions nos. KY305293, KY305294, KY305295, KY305296, and KY305298, respectively). Phylogenetic analysis of cloned sequences revealed the clustering of these putative aquaporins into two groups with human aquaporins (hAQPs): DerpAQP3 was clustered with hAQP8, hAQP11, and hAQP12; and DerpAQP1, DerpAQP2, DerpAQP4, DerpAQP5 were clustered with hAQP3, hAQP7 hAQP9, and hAQP10. This study provides a foundation for future studies of the importance of these proteins to dust mite functions and allergenicity.
We report the cloning and molecular characterization of a full-length cDNA encoding house dust mi... more We report the cloning and molecular characterization of a full-length cDNA encoding house dust mite allergen, Der f 6 from D. farinae isolated in China. The full-length Der f 6 cDNA was obtained with 840 nucleotides long. Nucleotide sequencing analyses showed a total of 36 mutations in five Der f 6 cDNA clones, corresponding to 23 incompatible amino acid residues. Recombinant Der f 6 (rDer f 6) protein was successfully expressed in and purified from E. coli BL21. Among 20 asthmatic patients, 45% was positive to rDer f 6 by ELISA. Bioinformatics analyses revealed that the mature Der f 6 was a hydrophobic and extracellular protein with chymotrypsin-like serine protease activity, its secondary structure was composed of alpha helix (7.69%), extended strand (34.62%), random coils (57.69%), and the similarity of Der f 6 to Blo t 6, Sui m 6, Der f 3 and Der f 9 was 64, 65, 35, and 38%, respectively.
International journal of biological macromolecules, 2023
Blomia tropicalis is an important species of allergenic mite. Structurally related cross-reactive... more Blomia tropicalis is an important species of allergenic mite. Structurally related cross-reactive allergens are involved in pathogenesis of clinical symptoms. The present study focused on recombinant allergen rBlo t 13 from B. tropicalis, including investigation of its structure, immunological properties, IgE-mediated cross-reactivity. In this work, the prokaryotic expression plasmids pET-28(a)-Blo t 13, pET-28(a)-Der f 13, and pET-28(a)-Tyr p 13 were constructed, transformed into E. coli Rosetta (DE3) pLysS, and purified by nickel affinity chromatography, respectively. By using ELISA, the IgE-binding rates were detected for rBlo t 13 and its epitope peptides, as well as the cross-reactivity among rBlo t 13, rDer f 13, and rTyr p 13. The tertiary structure of rBlo t 13 was resolved using X-ray diffraction at 2.0 Å resolution. Using IgE-ELISA, the IgE binding rate of rBlo t 13 was 60 % with Blomia tropicalis-positive sera. In the experiments of ELISA for cross-reactivity with rBlo t 13 on solid phase, the inhibition rates were 65 %, 57 % and 63 % for rBlo t 13, rDer f 13, and rTyr p 13, respectively. The structure of Blo t 13 protein contains a β-barrel structure which is composed of 10 β strands and has 2 α helices at the end of the barrel. Comparison of the tertiary structures of rBlo t 13, rDer f 13, and rTyr p 13 revealed that the β-barrel structure is highly conserved, consistent with the alignment of amino acid sequences. We obtained the recombinant protein rBlo t 13, demonstrated its cross-reactivity with Der f 13 and Tyr p 13 due to their structural similarity.
Dermatophagoides farinae, or the American house dust mite, is a common cause of allergy and asthm... more Dermatophagoides farinae, or the American house dust mite, is a common cause of allergy and asthma. Current tests for sensitization to D. farinae include an indirect enzyme-linked immunosorbent assay (ELISA) method for specific IgE detection, which, while clinically useful, is time-consuming and has low sensitivity since it uses crude mite extracts. We developed two new ELISA methods to detect the group 2 allergen from D. farinae (Der f 2) and the Der f 2-specific IgE in sera of patients with asthma. Using recombinant Der f 2 protein for the analysis of Der f 2-specific IgE, we tested both indirect ELISA and avidin biotin complex ELISA (ABC-ELISA) methods in 46 patients who were also tested by Pharmacia UniCap. Both of these approaches are more specific than traditional methods using crude mite extracts. These new tests could aid in the laboratory diagnosis of asthma due to sensitization to D. farinae.
Magnetic Fe3O4 nanoparticles (MNPs) have shown promise as drug carriers for treating lung and liv... more Magnetic Fe3O4 nanoparticles (MNPs) have shown promise as drug carriers for treating lung and liver tumors in vivo. However, little is known about the combined delivery of these MNPs with a second approach, extremely low frequency electro-magnetic field (ELFF) exposure, which has been shown to have value for in vitro treatment of tumor cells. Here, ELFF and MNPs were combined to treat healthy (HL-7702) and cancerous (Bel-7402, HepG2) hepatic cells lines to explore the potential therapeutic effects, bio-mechanisms, and potential toxicity of a combined drug-free treatment in vitro. Flow cytometry for anti-AFP (alpha fetal protein) antibody, which coated the MNPs, indicated that the combined treatment induced Bel-7402 and HepG2 hepatoma cells lines into early apoptosis, without significant effects on healthy hepatic cells. This effect appeared to be mediated through cellular membrane ion metabolism. The presence of AFP-loaded MNPs strengthened the effects of ELFF on tumor cells, inducing a higher frequency of early apoptosis, while having minimal toxic effects on healthy HL-7702 cells. Western blotting revealed that the apoptosis-triggering BCL proteins were up regulated in hepatoma cells compared to healthy cells. Flow cytometry and patch-clamp studies revealed that this resulted from a higher MNP uptake ratio and greater cellular membrane ion exchange current in tumor cells compared to HL-7702 cells. Further, patch-clamp results showed that combining MNPs with ELFF treatment induces cells into early apoptosis through an ion metabolism disturbance in cells, similar to ELFF treatment. In brief, the combination of ELFF and MNPs had beneficial effects on tumor cells without significant toxicity on healthy cells, and these effects were associated with cellular MNP uptake.
Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 2022
BACKGROUND:Dust mite extract contains multiple components that, while useful in clinical allergy ... more BACKGROUND:Dust mite extract contains multiple components that, while useful in clinical allergy diagnosis and treatment, can cause serious side effects. Defining components of dust mite extract is important their contributions to allergic disease. This study aimed to characterize a novel dust mite allergen, Der p 22. METHODS:We amplified the cDNA encoding Der p 22 from total RNA of the mite Dermatophagoides pteronyssinus, and inserted it into an expression construct for transformation to competent cells. Purified recombinant (r) Der p 22 was tested for IgE-binding reactivity in sera obtained from children with allergic asthma by the Affiliated Wuxi Children's Hospital of Nanjing Medical University (Jiangsu, China). rDer p 22 also was used to challenge BALB/c mice to assess effects on T helper cells and cytokine levels and applied to cultured lung epithelial cells to evaluate apoptosis and cytokine secretion. RESULTS:rDer p 22 bound to IgE in 93.75% of sera from pediatric allergic asthma patients. Mice challenged with rDer p 22 had altered Th1/Th2 ratios in spleen and lymph, and lower levels of cytokines IFN-γ but higher levels of IL-4 and IL-10 in alveolar lavage fluid compared with controls (p < .05). Cultured lung epithelial cells had greater apoptosis rates and exhibited higher levels of IL-6, IL-8, and GM-CSF when treated with rDer p 22 compared with control treatment (p < .05). CONCLUSIONS:Recombinant Der p 22 exhibited high IgE-binding rates in allergic children, indicating the activity of the recombinant protein and suggesting this novel allergen may be appropriate for inclusion in an allergy diagnostic workup. This finding is supported by in vitro and mouse in vivo studies showing rDer p 22 induced strong allergenic reactivity and apoptosis.
BACKGROUND:A clinical history of allergic symptoms and a skin-prick test with house-dust mite cru... more BACKGROUND:A clinical history of allergic symptoms and a skin-prick test with house-dust mite crude extracts are standard diagnostic procedures for Dermatophagoides pteronyssinus allergy. Specific immunoglobulin E (IgE) responses to Der p 1 and Der p 2 allergens have been used for the diagnosis of D. pteronyssinus allergy; however, evaluation of the diagnostic performance of Der p 1 and Der p 2 specific IgE (sIgE) produced inconsistent findings. We sought to evaluate the diagnostic accuracy of Der p 1 sIgE and Der p 2 sIgE measurement in the diagnosis of D. pteronyssinus allergy by performing a systematic review and meta-analysis of previously published studies. METHODS:Several medical literature electronic data bases were searched for related literature published through August 1, 2016. A bivariate model was used to pool estimates of sensitivity, specificity, diagnostic odds ratio, and area under the summary receiver operating curves as the main diagnostic measures. RESULTS:Eight studies, which involved 1095 patients, were included in our analysis. The pooled estimates of sensitivity, specificity, and diagnostic odds ratio for Der p 1 were 0.84, 0.97, and 166.57, respectively. The combined results for Der p 2 were a sensitivity of 0.87, specificity of 1.00, and a diagnostic odds ratio of 17342.35. The areas under the summary receiver operating curves for Der p 1 sIgE and Der p 2 sIgE were 0.94 and 0.98, respectively. CONCLUSION:Our results supported the use of Der p 1 and Der p 2 sIgE in the diagnosis of D. pteronyssinus allergy. Both displayed good diagnostic performance and would be useful in a clinical setting in the accurate diagnosis of dust mite allergy.
Objective:Diabetes is a growing global public health concern with many significant disease compli... more Objective:Diabetes is a growing global public health concern with many significant disease complications. Multiple studies show that bone turnover markers (BTMs) are decreased in diabetes patients, indicating impaired bone metabolism in diabetes patients. A recent study also showed that in diabetes patients, BTMs are correlated with urine albumin to creatinine ratio, an indicator of nephropathy. However, whether BTMs are correlated with estimated glomerular filtration rate (eGFR) in diabetes remains unknown. This retrospective study accessed correlations between serum BTMs and eGFR in Chinese patients with diabetes and compare levels of BTMs and eGFR between diabetic patients and healthy individuals. Methods:This study analyzed data from 221 diabetic patients (include type1 and type 2 diabetes) and 155 healthy individuals. Serum BTM levels and eGFR were compared between diabetic patients and healthy individuals. Pearson correlation analysis was used to assess correlations between BTMs and eGFR. Multiple logistic regression analysis adjusted for gender and age was performed to measure odd ratio (OR) and 95% confidence interval (95% CI) of BTMs on diabetes. Results:Patients with diabetes had significant lower 25-hydroxyvitamin D (25(OH)D) levels (15.07 ± 6.20 ng/mL) than healthy group (17.89 ± 6.41 ng/mL) ( < 0.05). For patients with diabetes, eGFR was negatively correlated with osteocalcin (OC) ( = -0.434, < 0.05), procollagen type 1 intact N-terminal propeptide (P1NP) ( = -0.350, < 0.05), and -carboxy-terminal cross-linking telopeptide of type I collagen (-CTX) ( = -0.179, < 0.05) levels. For healthy people, eGFR was negatively correlated with 25(OH)D ( = -0.290, < 0.05) levels. Multiple logistic regression analysis adjusted for age and gender (mean age of diabetes was 64.9 years and the percentage of female was 66.9%, mean age of healthy people was 48.4 years and the percentage of female was 37.4%) showed that 25(OH)D (OR = 0.909, 95%CI = 0.862 - 0.959, < 0.05) was protective factors for diabetes. Conclusions:In the stage of diabetic nephropathy, bone turnover may accelerate. It is important to detect BTMs in the stage of diabetic nephropathy.
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Papers by Yubao B Cui
Dermatophagoides (D.) pteronyssinus and investigate their ability to
cross-react with other group-29 allergens from D. pteronyssinus as
well as those from D. farinae and Tyrophagus putrescentiae.
Methods: Der p 29, Der f 29, and Tyr p 29 cDNA sequences were
amplified from total RNA isolated from D. pteronyssinus, D. farinae
and Tyrophagus putrescentiae, respectively. Then they were cloned
into the pET28a vector, expressed in Rosetta2(DE3)plysS, and
purified using anion exchange chromatography. The IgE-binding
rates of rDer p 29 were assessed by IgE Western blotting. The four
epitopes of rDer p 29 were predicted, synthesized, and detected
by IgE-ELISA. The cross-reactivity among the recombinant
proteins rDer p 29, rDer f 29, and rTyr p 29 was investigated using
dot blot and IgE-ELISA inhibition experiments. The allergens’
physiochemical properties, amino acid sequences, and tertiary
structures were also compared.
Results: Der p 29 was successfully expressed in Rosetta2(DE3)plysS
as a single, 393-bp open reading frame. Western blotting showed that
the purified rDer p 29 protein exhibited an IgE-binding rate of 100%
when tested on patient sera. The following four Der p 29 epitopes
were predicted and synthesized: 37-45 (EP1), 57-69 (EP2), 75-80
(EP3), and 104-117 (EP4). IgE-ELISA tests on 20 D. pteronyssinuspositive sera yielded IgE-binding rates of 85% (rDer p 29), 80%
(EP1), 55% (EP2), 40% (EP3), and 55% (EP4), respectively. The
dot blot experiments further confirmed cross-reactivity among
the three group-29 proteins. When used as an inhibitor, rDer p 29
demonstrated an average cross-reactive inhibition rate of 49.7%
against rDer f 29 and 54.4% against rTyr p 29. When rTyr p 29 was used as an inhibitor, it showed an average cross-reactive inhibition
rate of 56.3% against rDer f 29.
Conclusions: A recombinant protein, rDer p 29 with strong
allergenicity was produced. Moreover, it was found that rDer p
29 cross-reacted with rDer f 29 and rTyr p 29, due to their highly
homologous sequences and structures. These findings highlight the
importance of considering inter-species epitope cross-reactivity
when diagnosing and treating allergic diseases.
engineering. The CRISPR/Cas system provides a novel tool for manipulating genomic DNA using an approach that is simple, convenient, and easy to learn. Since its first description as a genome editor in 2012, it has been widely applied to bacteria, yeast, nematodes, flies, zebrafish, mice, rats, and human cells. This review describes the basic principles of the system and its application. In particular, we focus on CRISPR as a tool to manipulate and study the functional genomes of the planet’s most
abundant organisms, insects, which have diverse impacts on humans and the environment.
to study the transcriptome of the house dust mite Dermatophagoides pteronyssinus to identify putative aquaporin family members. RNA was isolated from more than 2000 mites for 5΄-RACE and 3΄-RACE. Putative aquaporins were identified through BLAST alignments, and primers were designed to amplify the five identified sequences. These cDNA fragments
were then inserted into bacterial plasmids for molecular cloning and sequencing of DerpAQP1, DerpAQP2, DerpAQP3, DerpAQP4, and DerpAQP5 (GenBank accessions nos. KY305293, KY305294, KY305295, KY305296, and KY305298, respectively). Phylogenetic analysis of cloned sequences revealed the clustering of these putative aquaporins into two
groups with human aquaporins (hAQPs): DerpAQP3 was clustered with hAQP8, hAQP11, and hAQP12; and DerpAQP1, DerpAQP2, DerpAQP4, DerpAQP5 were clustered with hAQP3, hAQP7 hAQP9, and hAQP10. This study provides a foundation for future studies of the importance of these proteins to dust mite functions and allergenicity.