Preparation of monoclonal antibodies against recombinant Der f 7 allergen / 中国免疫学杂志

Objective:To clone and express the Der f 7 recombinant protein from Dermatophagoides farinae and prepare the Der f 7 monoclonal antibody.Methods: The Der f 7 recombinant protein was expressed in prokaryotic expression system of pET28a(+)-Der f 7.BALB /c mice were immunized with the recombinant protein.Myeloma cells and spleen cells were fused,and hybridoma cells were screened by ELISA.Hybridoma cells were injected into the mice abdominal cavity to obtain ascites.It was purified by protein A agarose medium ascites,and then to dentified the titer and purity of the antibody.The specificity of the antibody was identified by Western blot.Results: Three hybridoma cells which stably secret recombinant Der f 7 monoclonal antibody were obtained.The monoclonal antibody had high purity,the titer was higher than 1∶243 000.Western blot showed that Der f 7 recombinant protein could be recognized well.Conclusion: We successfully obtained Der f 7 monoclonal antibody,which can be used for the quantification and localization of Der f 7 allergen and the diagnosis and treatment of allergic diseases.

Study on correlation between ERα gene PvuⅡ ,XbaⅠ and Erβ gene RsaⅠ , AluⅠ digestion polymorphism with coronary heart disease / 国际检验医学杂志

Objective To investigate the correlation of ERα gene PvuⅡ ,XbaⅠ and ERβ gene RsaⅠ ,AluⅠ digestion polymorphism with coronary atherosclerotic heart disease(CHD) risk factors in Yancheng area .Methods A total of 124 cases of CHD and 163 persons undergoing physical examination served as the CHD group and CON group .The enzyme method was adopted to detect TG and TC .The direction method was adopted to detect HDL and LDL .ERα gene PvuⅡ ,XbaⅠ and ERβ gene RsaⅠ ,AluⅠ digestion polymorphisms were detected by adopting RFLP-PCR .Results The ratios of smoking history ,family history ,complicating hypertension and diabetes ,and the level of body mass index ,TC ,TG and LDLC in the CHD group were significantly higher than those in the control group ,the difference was statistically significant (P＜0 .05) .The various indicators had no statistically difference between male and female(P＞0 .05) .The frequency distribution and geographic distribution of ERα gene PvuⅡ ,XbaⅠ and ERβ gene RsaⅠ ,Alu Ⅰ digestion polymorphisms had no difference between the two groups ,all conformed to Hardy-Weinberg genetic equilibrium and had the group representativeness .pp ,xx ,RR and AA genotypes in the CHD group were maximal ,while PP , XX ,rr and aa genotypes were minimal ;Pp ,xx ,RR and AA genotypes in the CON group were maximal ,while PP ,XX ,rr and aa genotypes were minimal .The distribution frequency of p and x genes in the CHD group was significantly higher than that in the control group ,the difference was statistically significant(P＜0 .05) .Conclusion The estrogen gene polymorphism might be a target spot for effectively treating CHD ,and p and x gene distribution frequency may be related with CHD risk factors .

Structure and func tion comparation of dust mite allergen Der f 3,Der p 3 and Eur m 3 / 中国免疫学杂志

Objective:To compare structure and function of mite Der f 3,Der p 3 and Eur m 3.Methods: Obtained mite allergens amino acid sequences from the International Union of Immunological Societies nomenclature database .Then physiochemical characterization,sequence alignment,secondary structure,three dimensional (3D) structure and epitopes of three proteins were analyzed by bioinformatics methods.Results:According to results of alignment ,Der f 3,Der p 3 and Eur m 3 displayed 88.51%sequence iden-tity.Der f 3,Der p 3 and Eur m 3 all contained three active sites and two trypsin functional domains ,which showed high identity of amino acid residues.Active sites of three proteins ,which closing to each other in three dimensional (3D) structure,constituting the active center of the enzyme.Secondary and 3D structure of three proteins all contains α-helices,β-sheets and random coils.Epitopes analysis revealed that Der f 3,Der p 3 and Eur m 3 all have 5 main potential epitopes located in random coils.Epitope sequences of Der f 3,Der p 3 and Eur m 3 overlapping in three domains (peptides of 79-81aa,129-135aa and 172-174aa),but the residues in these three domains were not identical.Conclusion:These studies lay the foundation for biochemical and genetic analysis of these 3 allergens,and may contribute to vaccine development for allergen-specific immunotherapy.