Study On Cloning, Expression And Immunological Characteristics Of Dermatophagoides Farinae HSP60

At present, the allergic diseases have become a global public health problem.Dust mites widely present in the indoor environment, as an important allergen, theyare highly valued in allergological research. Dust mites can cause bronchial asthma,allergic rhinitis, allergic dermatitis and other allergic diseases, and take a seriousimpact on human health. The research of dust mite allergens (dust mite componentsrelated to allergy) has been paid close attention. With further exploration, new dustmite allergens were identified and found, it greatly deepened the humanunderstanding about dust mite allergens and promoted the level of diagnosis,prevention and treatment of dust mite allergic diseases. The heat shock protein60(Hsp60) from parasites usually has a good immunogenicity. Therefore, maybe it hasthe potential ability that can induce the host’s allergic reactions and become a dustmite allergen. On the basis of our previous study, this research choosed theDermatophagoides farinae(D.f) Hsp60as the research object, its cDNA sequence wasdetected. Meanwhile, the Hsp60gene fragment was cloned and transformed into E.coli for prokaryotic expression, the immunological characteristics of the expressionproduct was analysed.Objective:The D. f Hsp60as the research object, measuring and analyzing its cDNAsequence, then it was cloned into a vector for expressing of Hsp60protein fragments.With D. f allergic patients’ serum as probe, the immunological characteristics of the recombinant protein was primarily assessed. Our aim was to understand if Hsp60is anew D. f allergens.Methods:1、The total RNA of D. f was extracted with Trizol.2、The cDNA synthesis and PCR amplification of D. f The RNA extracted astemplate, cDNA was synthesized by reverse transcription; on basis of Hsp60’s thecharacteristic and conservative sequence of the species close to D. f, thedegenerate primers were designed, the cDNA as a template for the PCRamplification of Hsp60gene fragment.3、Sequencing and amino acid sequence analysis The amplified PCR products weresequenced; The sequences of the cDNA and the amino acid sequence encoded byit were determined, they were analyzed in homology and the hydrophilic regionsof amino acid sequence were understood.4、Amplification and TA cloning of D. f Hsp60gene fragments Specific PCRprimers were designed by the sequencing results and restriction sites added in theprimers. Hsp60gene fragments of D. f were amplified by PCR, and then wereconnected to the PMD18-Tvector, and positive recombinant plasmids weresequenced.5、Subcloning of D. f Hsp60gene fragments The TA cloning plasmids andpET-32a(+) plasmids were digested by BamHⅠand XhoⅠ,and then the aim genefragment were connected on pET-32a(+) vector, building a pET-32a(+)-Hsp60prokaryotic expression vector.6、Expression and purification of recombinant protein The positive recombinantplasmids were transformed into E.coli BL21and the recombinant protein wasexpressed by the bacteria under induction of IPTG. The express products wereobserved and analyzed by SDS-PAGE. The expressed recombinant protein waspurified by Ni-NTA column.7、Western blot analysis of recombinant protein The D. f allergic patients’ sera wereused to react with the recombinant proteins, Goat Anti human IgE-HRP as a secondary antibody，finally the results of Western blot were showed by ECLchemiluminescence method.8、Spectral analysis of recombinant protein The bands of purified recombinantprotein in SDS-PAGE were sent to Guangzhou Huijun company for spectralanalysis.Results:1、 D. f RNA extracted as template, the cDNA were synthesized by reversetranscription. The products about520bp were successfully amplified through thedegenerate primers PCR. Sequencing results showed that the nucleotide sequencehad similarity of86%with Hsp60of bacterium Achromobacter Xylosoxidans, theencoded amino acid sequence similarity of92%with Advenella MimigardefordensisHsp60.2、 The recombinant prokaryotic expression vector (pET-32a(+)-Hsp60) wassuccessfully constructed，and the recombinant protein about37KD was expressed,the soluble recombinant proteins were obtained by Ni-NTA purified column.3、The purified recombinant protein were analyzed by Western blot method. Theresults showed it may be identified by the IgE in D. f allergic patients’ sera.4、The result of spectrometry analysis showed that the recombinant protein is Hsp60.Conclusions:1、Combining immunoblotting, proteomic analysis and degenerate primers PCRtechnology may be a kind of effective method in screening new dust miteallergens, it provides a new technical route for discovery of new dust miteallergens.2、The partial cDNA sequence of D. f Hsp60was first determined by the methods ofthe degenerate primers PCR and DNA sequencing.3、The recombinant protein of D. f Hsp60was first obtained, and it was confirmed thatthe recombinant protein can react with the IgE in D. f allergic patients’ sera. Inthis study, the important evidence that Hsp60may be a new allergen of D. f has been acquired.