Cloning, expression,purification and immunological identification of Der f 11, paramyosin,a recombinant allergen of dust mite / 中国免疫学杂志

Objective: To clone , express and purify of Dermatophagoides farinae ( Der f 11 ) , and then test its immunogenicity.Methods:The gene coding for Der f 11 was synthesized ,and was then linked with the pET-32a vector.The expression plasmid pET32a(+)-Der f 11 was induced by IPTG.After purification of recombinant allergens Der 11 proteins through the Ni +affinity chromatography ,immunological allergic patients serum as the Primary antibody.Results: We obtain high purity recombinant Der f 11 protein.The results of SDS-PAGE show that the expression product is about 118 KD.Recombinant allergen Der f 11 test 15 dust mites allergic patients serum specific IgE , positive rate was 20%.Conclusion: Recombinant allergen Der f 11 obtained has the similar immunologic activity to natural Der f 11 protein.It can lay the foundation for the specific diagnosis ,treatment and further experimental studies of the dust mite allergy disease.

Clinical Value of Detecting Serum GP7 3 and Related Indexes of Liver Fibrosis Levels in Patients with Hepatitis B Cirrhosis / 现代检验医学杂志

Objective To investigate the relationship between serum Golgi protein-73 (GP73)level and related indexes of he-patic fibrosis in patients with hepatitis B cirrhosis diseases and discuss the clinical value of GP73.Methods The level of GP73 was detected by ELISA;the levels of type Ⅳ precollagen (Ⅳ-C)and hyaluronic acid (HA)were measured by radio-immunity in serum from 11 patients with acute hepatitis B (AHB),31 patients with chronic hepatitis B (CHB),71 patients with Hepatitis B Cirrhosis (LC),23 patients with primary hepatic carcinoma (PHC)and 34 healthy people with check-up. The clinical changes and relationship between the GP-73 level and the parameter of fibrous degeneration were studied.Re-sults The GP73 level in CHB group was 118.0±82.2μg/L;the GP73 level in LC group was 154.4±87.7μg/L and the GP73 level in PHC group was 196.5±89.6μg/L.Compared with the normal control group,statistically significant differ-ence were found in GP73 level of CHB group,LC group and PHC group (t values were -7.284,-7.909 and -9.609,re-spectively;P<0.01).As the progress of liver cirrhosis,the levels of GP73,Ⅳ-C and HA all increased gradually and the level of GP73 was significantly positively correlated to Ⅳ-C and HA (r valves were -0.212 and 0.487,respectively;P<0.01). Conclusion The GP73 was high expressed in chronic liver disease,especially in liver cirrhosis.The GP73 was significantly associated with the occurrence of hepatitis b liver fibrosis.There fore,GP73 could be used as a better index to monitor the fi-brosis degree.

Gene cloning,prokaryotic vector constructing for the major allergen ovomucoid in Hen's egg white and its recombinant expression / 中国免疫学杂志

Objective:To clone and express the gene of ovomucoid,which is the main allevgen in egg white.Methods:Using total RNA of chicken oviduct as template,the gene of ovomucoid was amplified by RT-PCR.The homology was analyzed by comparision with the sequence in GeneBank.Subsequently,the PCR product of the ovomucoid gene was cloned into prokaryotic expressing vector pET-28a and was expressed by the challenge of IPTG.Results:The whole gene of ovomucoid,one of the main allergens in egg white,was successfully cloned.The cloned ORF sequence contains 633 bp,including stop codon,encods for 210 amino acids.Sequence analysis shows that the ovomucoid gene displays 99% nucleotide identities with the published sequences.The molecule weight of ovomucoid protein obtained was 21 kD.By the challenge of IPTG,SDS-PAGE analysis showed that the ovomucoid gene was overexpressed in E.coli BL21(DE3).Conclusion:The gene of ovomucoid is cloned and overexpressed in E.coli BL21(DE3).This study will be the basis for the further research.

Cloning,expression and the immune characterization of panallergen profilin in coco(Cocos nucifera Linnaeus) / 中国免疫学杂志

Objective:To clone and express panallergen profilin from the pollen of coco(Cocos nucifera Linnaeus).Methods:RT-PCR and RACE methods were applied to clone the full-length panallergen genes from coco pollen and the sequence was analyzed.The specific primers were designed.The ORF of profilin of coco pollen was amplified with RT-PCR and cloned into the expression vector pET 28a.Expression of the recombinant coco pollen profilin was carried out in E.coli BL21(DE3) and the purification of the recombinant protein was performed via affinity chromatography with Ni2+ coupled to sepharose.IgE reactivity to recombinant coco pollen profilin was investigated by immunoblot.Results:The complete sequence of coco pollen profilin was cloned.The sequence was 608 bp and included an open reading frame(396 bp) coding for 131 amino acids.Sequence analysis showed that the deduced protein was an acidic protein with an estimated molecular mass of 14.19 kD and a pI of 4.61.The GeneBank accession number of the clones was EF173598.After overexpressed in E.coli BL21(DE3),the recombinant protein was purified through affinity chromatography with Ni2+ coupled to sepharose.Immunoassay showed that the recombinant allergen has good IgE binding capacity.Conclusion:The profilin of coco pollen is expressed successfully in BL21(DE3),which will be used as a base for further study on coco pollen related allergy.