Expression of miR-155 in Serum Exosomes in Children with Epilepsy and Its Diagnostic Value.

Objective: This study was designed to analyze the expression of miR-155 in serum exosomes in children with epilepsy and to explore its diagnostic value. Methods: From March 2020 to March 2021, 43 hospitalized children with epilepsy admitted to the Department of Neurology of the hospital were included, and another 43 gender- and age-matched healthy children were randomly selected as the healthy control group during the same period. Then fasting serum samples of the two groups were collected to extract the exosomes. The morphology of the exosomes was evaluated under a transmission electron microscope, and the expression of specific protein markers on the surface was detected by Western Blot. In addition, the relative expression of miR-155 in serum exosomes in children with epilepsy with different courses of the disease and different degrees of abnormal electroencephalography (EEG) was compared, and the area under the receiver operating characteristic (ROC) curve (ROC-AUC) was used to evaluate the diagnostic value of miR-155. Results: A higher relative expression level of miR-155 in serum exosomes was obtained in the epilepsy group, as compared to the healthy control group (P<0.05), and the relative expression of miR-155 in serum exosomes in children with epilepsy was correlated with the course of the disease and the degree of abnormal EEG (both P<0.05). In addition, the expression of miR-155 in serum exosomes showed high diagnostic efficiency for epilepsy (AUC = 0.813, P<0.05). Conclusion: The expression of miR-155 in serum exosomes in children with epilepsy is up-regulated, and its level is related to the course of the disease and the degree of abnormal EEG, so miR-155 in serum exosomes may be used as a biomarker for the diagnosis and assessment of the severity of epilepsy.

Two allergens from Scylla paramamosain share common epitopes showed different allergenic potential in Balb/c mice.

Filamin C (FLN c) and triosephosphate isomerase (TIM) are novel allergens of crab (Scylla paramamosain) which are sharing common epitopes. This work aimed to assess their contributions to the induction and elicitation of allergenic responses. Balb/c mice were sensitized by intraperitoneal injections and challenged by intragastric gavage with purified proteins. Upon oral challenge, FLN c triggered more severe anaphylactic symptoms, higher levels of specific antibodies and histamine in serum than TIM, while TIM was a more active promotor of early specific antibody production and stimulated stronger Th2-biased responses. Combined with the results of in vitro assays, the data demonstrated that though with common epitopes, the two allergens showed a different allergenicity, TIM favored Th2 polarization in sensitization stage, while FLN c had a better ability to stimulate B cells and is highly immunogenic in oral challenge stage. The findings can help with the better understanding of allergenicity of crab allergens.

Cloning, expression and comparison of the properties of Scy p 9, a Scylla paramamosain allergen.

This study investigated the properties of Scy p 9 in mud crab (Scylla paramamosain). The gene sequence of filamin C obtained from crabs, which was denoted as Scy p 9, contains a 2544 bp open reading frame and encodes 848 amino acid residues. Recombinant Scy p 9 (rScy p 9) is expressed in Escherichia coli, which exhibits tertiary structure changes, and the IgE binding activity of rScy p 9 is higher than that of native Scy p 9 (nScy p 9). Moreover, this study explored the possibility of the presence and cross-reactivity of filamin C in 8 shellfish. IgE-specific binding to nScy p 9 and rScy p 9 in patients allergic to shellfish revealed that rScy p 9 was more sensitive than nScy p 9. The gene sequence of filamin C fills in the blank in shellfish. This study contributes to the understanding of the properties of Scy p 9, and the results indicate that rScy p 9 can be used as a candidate for component-resolved diagnosis in shellfish.

Expression and epitope identification of myosin light chain isoform 1, an allergen in Procambarus clarkii.

Myosin light chain isoform 1 (MLC1) is reported to be a novel allergen in crayfish (Procambarus clarkii). However, little information is available about its allergic epitopes. In this study, recombinant crayfish MLC1 (rMLC1) was expressed and confirmed by mass spectrometry. Circular dichroic analysis and serological test were performed for the measuring of structural and immunological properties of rMLC1. Specific-protein-A-enriched IgG raised in rabbits against purified rMLC1 was used to screen a phage display random peptide library. Nine MLC1 mimotope clones were identified among 16 random clones after biopanning. Five conformational epitopes were identified with the program LocaPep, and mapped into 3 epitope regions at the antibody-binding interface of MLC1. MLC1 of crayfish showed high primary and secondary structure identity to MLC of other allergenic species, its epitopes were located in the structure conserved regions, and its cross-reactivity among related species was indicated by immunological assays.

A comprehensive analysis of the allergenicity and IgE epitopes of myosinogen allergens in Scylla paramamosain.

BACKGROUND: Scylla paramamosain is one of the most common and serious food allergens in Asia. Therefore, research on its prevalence, accurate diagnosis, and IgE-binding pattern of the allergens is crucial. OBJECTIVE: To identify the IgE epitopes of the myosinogen allergens in S. paramamosain using phage peptide library. METHODS: The prevalence of allergy to crabs (AC) and of sensitization was analysed using a questionnaire and a serological assay. BAT was performed by flow cytometry, and its diagnostic performance was evaluated in relation to allergens purified from crab myosinogen. IgE-binding epitopes were identified by phage display using the IgE from patients with AC. Sequence- and structure-based bioinformatics analyses were performed to identify allergenic epitopes. RESULTS: Crab was the most common cause of food allergies in this study. Subjects with AC (n = 30) with clear clinical symptoms were identified by immunoblotting and BAT. All of the myosinogen allergens triggered basophil activation; surface expression of CD63 and CD203c was higher in patients allergic to AK and FLN c than in patients allergic to SCP and TIM. In addition to six conformational epitopes of SCP, six linear epitopes and eight conformational epitopes of AK were identified. Five linear epitopes and three conformational epitopes of TIM, nine linear and ten conformational epitopes of FLN c were also identified, and the sequence VH(I/T) L was appeared in epitopes of both TIM and FLN c. The number of epitopes showed consistency with the value of BAT. CONCLUSIONS AND CLINICAL RELEVANCE: BAT can be used for accurate diagnosis of AC. Identification of particular allergenic motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergies.

Genome- Wide Analysis of the Nucleotide Binding Site Leucine-Rich Repeat Genes of Four Orchids Revealed Extremely Low Numbers of Disease Resistance Genes.

Orchids are one of the most diverse flowering plant families, yet possibly maintain the smallest number of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) type plant resistance (R) genes among the angiosperms. In this study, a genome-wide search in four orchid taxa identified 186 NBS-LRR genes. Furthermore, 214 NBS-LRR genes were identified from seven orchid transcriptomes. A phylogenetic analysis recovered 30 ancestral lineages (29 CNL and one RNL), far fewer than other angiosperm families. From the genetics aspect, the relatively low number of ancestral R genes is unlikely to explain the low number of R genes in orchids alone, as historical gene loss and scarce gene duplication has continuously occurred, which also contributes to the low number of R genes. Due to recent sharp expansions, Phalaenopsis equestris and Dendrobium catenatum having 52 and 115 genes, respectively, and exhibited an "early shrinking to recent expanding" evolutionary pattern, while Gastrodia elata and Apostasia shenzhenica both exhibit a "consistently shrinking" evolutionary pattern and have retained only five and 14 NBS-LRR genes, respectively. RNL genes remain in extremely low numbers with only one or two copies per genome. Notably, all of the orchid RNL genes belong to the ADR1 lineage. A separate lineage, NRG1, was entirely absent and was likely lost in the common ancestor of all monocots. All of the TNL genes were absent as well, coincident with the RNL NRG1 lineage, which supports the previously proposed notion that a potential functional association between the TNL and RNL NRG1 genes.

De novo characterization of the Iris lactea var. chinensis transcriptome and an analysis of genes under cadmium or lead exposure.

Iris lactea var. chinensis (I. lactea var. chinensis) is tolerant to accumulations of cadmium (Cd) and lead (Pb). In this study, the transcriptome of I. lactea var. chinensis was investigated under Cd or Pb stresses. Using the gene ontology database, 31,974 unigenes were classified into biological process, cellular component and molecular function. In total, 13,132 unigenes were involved in enriched Encyclopedia of Genes and Genomes (KEGG) metabolic pathways, and the expression levels of 5904 unigenes were significantly changed after exposure to Cd or Pb stresses. Of these, 974 were co-up-regulated and 1281 were co-down-regulated under the two stresses. The transcriptome expression profiles of I. lactea var. chinensis under Cd or Pb stresses obtained in this study provided a resource for identifying common mechanisms in the detoxification of different heavy metals. Furthermore, the identified unigenes may be used for the genetic breeding of heavy-metal tolerant plants.

Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A71G74S69D75T73F72V67) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis.

Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Purification, characterization and immunoreactivity of ß'-component, a major allergen from the roe of large yellow croaker (Pseudosciaena crocea).

Fish roe, a nutritious food, is favored by consumers, but has also been confirmed to be allergenic in salmonid fish. However, little information is available in other fish species. To determine the allergen in the roe of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera of allergic patients. The major allergen was purified by column chromatography methods, revealing a single band with 16 kDa and was confirmed as ß'-component (ß'-c) by mass spectrometry. The results of physicochemical characterization showed that ß'-c was a glycoprotein and was relatively stable following thermal or acid/alkali treatment. Furthermore, ß'-c was easily degraded by pepsin, but was resistant to trypsin and α-chymotrypsin. After treatment with different processing methods, including Maillard reaction (MR), ultraviolet radiation (UVR), ultrasound-heat (UH), and retorting (RT), the IgG-binding activity of ß'-c decreased obviously by MR, but decreased slightly by UVR and UH. Cross-immunoreactivity results of the allergens in the roes of different species revealed that ß'-c was a specific allergen in teleostean, and the cross-immunoreactivity between the roe of large yellow croaker and other kinds of fish roe was relatively strong.

Free perforator flap transfer for reconstruction of skull base defects after resection of advanced recurrent tumor.

BACKGROUND: Skull base reconstruction is challenging due to its proximity to important anatomical structures. This report evaluates the use of perforator flaps for reconstruction of skull base defects after advanced recurrent tumor resection. METHODS: Fourteen free perforator flaps were transferred to reconstruct skull base defects in 14 consecutive patients, from October 2004 to May 2011. All patients had advanced recurrent neoplasms that were previously treated with either radiation therapy or surgery. The surgical defects were reconstructed using various perforator flaps mainly the deep inferior epigastric artery perforator flaps, anterolateral thigh (ALT) flaps, or thoracodorsal artery perforator flaps. The outcomes following reconstruction and associated complications were evaluated. RESULTS: The overall free flap success rate was 93% (13/14). One ALT flap was lost. Three patients (20%) had a cerebrospinal fluid fistula, and two of them developed meningitis. No complications were observed at the donor site. CONCLUSIONS: The use of perforator flaps may be a viable option for reconstruction of skull base defects after the resection of advanced recurrent tumor.

[Clinical analysis of 318 cases of oropharyngeal squamous cell carcinoma].

OBJECTIVE: To investigate the clinicopathological features, treatment outcomes and prognosis of patients with oropharyngeal squamous cell carcinoma (OSCC). METHODS: Retrospective review of 318 consecutive cases with OSCC treated from January 1999 to December 2011. Survival rates and prognostic factors were calculated using the Kaplan-Meier method and multivariate Cox model survival analysis respectively. There were 281 males and 37 females, with a median age of 56 years. Of the 318 cases, 163 carcinomas were from tonsil, 108 from tongue base and 47 from soft palate-uvula. The presenting symptoms were pharyngalgia (128 cases, 40.3%), neck masses (71 cases, 22.3%), foreign body sensation in the pharynx (63 cases, 19.8%) and dental ulcer (44 cases, 13.8%). The median time from onset of the first symptoms until diagnosis of OSCC was 3 months. Of the 318 OSCC, 75 were with high grade, 110 with intermediate grade and 133 with low grade, including 10 patients at stageI, 39 at stage II, 68 at stage III and 201 at stage IV. RESULTS: The rates of lymph node metastasis, distant metastasis and second primary carcinoma were 72.3%, 13.2% and 7.9%, respectively. Of 318 patients, 117 received radiotherapy alone, 66 underwent surgery plus postoperative radiotherapy, 59 underwent preoperative radiotherapy plus surgery, 33 received concomitant chemotherapy and radiotherapy, 20 received concomitant molecular targeted therapy and radiotherapy, 16 underwent surgery alone and 7 received induction chemotherapy plus radiotherapy. The 3-, 5-year overall survival (OS) rates were 58.4%, 50.7%, respectively, and the median overall survival time was 60.1 months. Ages (P = 0.034), gender(P = 0.024), smoking and alcohol consumptions(P = 0.008), doses of radiotherapy(P = 0.046) and clinic stages(P = 0.001) were independent factors for OS. CONCLUSIONS: OSCC is poor in prognosis, with a high incidence of cervical lymph node metastasis. Radiotherapy and salvage surgery are the main treatments for OSCC.

Purification, physicochemical and immunological characterization of arginine kinase, an allergen of crayfish (Procambarus clarkii).

Arginine kinase (AK) has attracted considerable attention because it has been identified as a shellfish allergen. However, little information is available about AK in crayfish (Procambarus clarkii). In this study, crayfish AK was purified and cloned. Its physicochemical properties, processing stability, and immunological characteristics were analyzed. Crayfish AK was purified by column chromatography, which revealed a single band with molecular mass of 40 kDa; this result was further confirmed by mass spectrometry. The full-length gene sequence of crayfish AK was 1462 bp and encoded a protein of 357 amino acid residues. The results of this study revealed that crayfish AK is a glycoprotein with an isoelectric point of approximately 6.5. Thermal stability assays revealed that crayfish AK easily forms aggregates at temperatures >44°C and was stable at pH 4.0-8.0. SDS-PAGE and dot blotting were used to assess processing stability of purified AK. The results revealed that the IgE-binding activity of crayfish AK is reduced after boiling.

[Primary squamous cell carcinoma of the thyroid: retrospective analysis of 28 cases].

OBJECTIVE: To investigate the clinicopathological features, treatment outcomes and prognosis of patients with squamous cell carcinoma of the thyroid (SCCT). METHODS: Retrospective review of SCCT cases in our hospital from January 1999 to May 2012. Demographic data and clinical charts, including presenting symptoms, histologic grade of tumor, treatment, and outcome of 28 consecutive patients were obtained. Survival rates and prognostic factors were calculated with SPSS 13.0 software using the Kaplan-Meier method and multivariate Cox model survival analysis. RESULTS: SCCT accounted for only 0.36% of all types of thyroid malignancy. There were 15 males and 13 females, and the median age was 63 years. The presenting symptoms were neck masses (26/28) and hoarse voice (18/28). The 28 SCCTs included 15 high grade tumors, eight intermediate grade tumors and five low grade tumors. According to the UICC 2002 staging criteria, 16 patients were stage IVa, and 12 were stage IVb. Of the 28 patients, 19 underwent surgery plus postoperative radiotherapy, seven underwent surgery alone, and two received radiotherapy alone. The rates of lymph node metastasis and distant metastasis were 60.7% and 25.0%, respectively. The 1-year, 2-year and 5-year overall survival (OS) rate were 50.4%, 25.8% and 19.3%, respectively, and the median overall survival time was 12.2 months. Kaplan-Meier univariate survival analyses indicated that the sizes of the tumors, esophageal invasions and treatment policies are prognostic factors, and multivariate Cox model survival analyses confirmed that the sizes of the tumors and treatment policies were independent factors for OS. Multivariate survival analyses confirmed that the sizes of the tumors and treatment policies were independent factors for OS. CONCLUSIONS: SCCT is a rare malignant tumor with strong invasive ability, high malignancy and poor prognosis. Combined modality therapy was strongly recommended, and surgical resection plus postoperative radiotherapy may be the main treatment protocol for patients with SCCT.

[Gene construction and screening of humanized single chain antibody library against VSTM1-v2 cytokine].

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

[Human papillomavirus infection in oropharyngeal squamous cell carcinoma and prognosis: a preliminary analysis of 66 cases].

OBJECTIVE: To analyze the relationship between the prognosis of patients with oropharyngeal squamous cell carcinoma (OSCC) and human papillomavirus (HPV) infection in OSCC. METHODS: Sixty-six patients with oropharyngeal carcinoma who met the enrollment criteria during the period from January 1999 to December 2009 were retrospectively reviewed. The presence or absence of HPV oncogenic types in OSCC specimen was determined by multiplex polymerase chain reaction (PCR). Overall survival (OS) and disease specific survival (DSS) for HPV-positive and HPV-negative patients were estimated by Kaplan-Meier analysis. Cox regression model was used for multivariate analysis. RESULTS: HPV-DNA was detected in 11(16.7%) of all specimens. Among them, 7 were positive for HPV-16, 1 for HPV-16/11, 1 for HPV-35, 1 for HPV-58/52, and 1 for HPV-33/52/54. With the follow-up of 3-78 months (a median of 24.5 months), patients with HPV-positive tumors had significantly better overall survival (χ2=5.792, P=0.016) and disease specific survival (χ2=4.721, P=0.030), the 3-year OS and DSS were 90.0% vs 52.4% and 90.0% vs 56.4%, respectively. Multivariate analysis by Cox regression model showed that HPV infection and nodal status were both independent prognostic factors for patients with OSCC (P<0.05). CONCLUSIONS: Patients with HPV-positive OSCC have significantly better prognosis than patients with HPV-negative tumors. HPV infection is an independent prognostic factor.

Human papillomavirus infection and prognostic predictors in patients with oropharyngeal squamous cell carcinoma.

This study focused on infection rates and subtypes of human papillomavirus (HPV) in patients with oropharyngeal squamous cell carcinoma (OSCC), and the relationship between HPV status and prognosis of the disease. We evaluated sixty-six OSCC patients who met the enrollment criteria during the period from January 1999 to December 2009. The presence or absence of oncogenic HPV types in tumors was determined using the SPF10 LiPA25 assay. Overall survival (OS) and disease specific survival (DSS) for HPV positive and HPV negative patients were estimated using Kaplan-Meier analysis. The Cox regression model was applied for multivariate analysis. HPV-DNA was detected in 11(16.7%) of all specimens. Among them, 7 were type HPV-16, while other types were HPV-16/11, HPV-35, HPV-58/52, and HPV-33/52/54. Patients with HPV positive tumors were more likely to be female, non-smokers and non-drinkers (p=0.002, 0.001 and 0.001, respectively). After a median follow-up of 24.5 months, patients with HPV positive tumors had significantly better overall survival (HR=0.106[95%CI=0.014-0.787], p=0.016,) and disease specific survival (HR=0.121[95%CI=0.016-0.906], p=0.030). Patients with HPV positive OSCC have significantly better prognosis than patients with HPV negative tumors. HPV infection is an independent prognostic factor.

[Analysis of arterial spin labeling in 33 patients with hypoxic ischemic encephalopathy].

OBJECTIVES: To evaluate the diagnostic value of arterial spin labeling (ASL) technology in newborns with hypoxic ischemic encephalopathy (HIE). METHOD: Seven full-term newborn infants without any history of asphyxia and other nervous system diseases were selected as the control and 33 full-term newborn infants were assigned into HIE group. The patients in HIE group were further divided into three subgroups (19 cases of mild, 6 cases of moderate and 8 cases of severe HIE) based on their clinical diagnosis. The control group and HIE group were examined with GE Signa EXCITE HD 3.0T superconducting MRI scanner with a head phase array coil. Both groups were scanned with conventional axial MRI (T1FLAIR, T2WI and T2FLAIR), 1HMRS (PRESS sequence) and ASL (FAIR). Original images of 1HMRS and ASL were processed by Functool software of ADW 4.3 workstation. ASL perfusion images were observed and the signal intensity values of the region of interest (bilateral gray, white matter and basal ganglia) of the two groups were quantitatively measured, and mean value were calculated and compared between groups. Statistical analysis was performed with SPSS 13.0 software, and statistically significant difference was set at P < 0.05. RESULT: The perfusion images of two groups were obtained perfectly. The signal intensity values of bilateral gray, white matter and basal ganglia of control group were 125.34 ± 11.76, 73.42 ± 11.67 and 173.65 ± 15.49, respectively and there was a statistically significant difference between the different areas. The signal intensity values of bilateral gray, white matter and basal ganglia of HIE group were 153.47 ± 11.72, 71.35 ± 10.37 and 217.13 ± 12.51, respectively. There was a statistically significant difference (P < 0.05) in the average signal intensity value of gray matter and basal ganglia, but there were no statistically significant difference (P > 0.05) in white matter between the two groups. CONCLUSION: ASL Perfusion technique can assess HIE comprehensively and accurately. Furthermore, it can evaluate the brain damage of hypoxic ischemia. The results provide a strong basis for clinical treatment.

[Ribosome display screening of a novel human anti-IgE scFv fragment].

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.