ABSTRACT
Background Blepharitis caused by Demodex infestation is very common in clinical practice.There are various methods mentioned in the study of Demodex infestation in China,but a unified introduction and evaluation of the operating procedures is lacked.A quick and accurate clinical diagnostic method for Demodex infestation needs to be further studied.Objective This study aimed to establish operation procedures for the clinical examination of eyelid Demodex infestation,which were applied to evaluate the conditions of eyelid Demodex infestation in ocular patients with discomfort.Methods One thousand and fifty-two patients with eye dryness,eye itchiness or other symptoms were selected for slit lamp examination and photographing of the eyelid margin area.Three eyelashes with associated scurf from each superior eyelid were plucked out for examination of Demodex under the microscope.Positive findings included observation of Demodex mites or eggs.Their amounts were recorded individually for all eyelash samples.Results A procedure for observing,recording and reporting eyelid Demodex infestations in patients was successfully established.By using this procedure,1 052 patients were investigated for the examination of Demodex infestations.Demodex mites or eggs were found in 582 cases (55.3％).The positive rate of Demodex infestation increased with age,and the population over 60 years group had the highest positive rate,showing a significant difference among the different age groups (x2=10.547,P=0.001).There was no significant difference in positive rate between male patients and female patients (P =0.352).The test turnaround time (TAT) for one examination was (11.4±5.2) seconds.Conclusions The operational procedure for examining the palpebral margin Demodex infestation by the slit-lamp,optical microscope,photographing and laboratory reports is established.It is simple and quick in the appliation for the clinical diagnosis of eyelid Demodex infestation.
ABSTRACT
Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.