Cloning And Expression Of Aspergillus Fumigatus Genes Hsp70 And Hyp1

Out of more than 100’000 known fungal species, only about 300 cause disease in humans, Although,our body temperature may provide a protective thermal barrier against the majority of species that grow best at ambient temperature. There are still fungi can resistant to high temperature, especially in immunocompromised or critically ill. The main pathogen is Candidas, but the infection caused by Aspergillus,Cryptococcus, etc. are increasing, especially Aspergillus. Worldwide there are 200000 estimated annual cases of invasive aspergillosis(IA). Although Aspergillus infection rate second only to Candidas, but the mortality rate is much higher than the candida infection, up to 40% ~ 90%. IA is caused mainly by Aspergillus fumigatus,accounting for 90% ~ 95%, due to its ubiquitous spores. The diagnosis of IA is challenging and requires a high level of suspicion. Due to the lack of fast and accurate diagnostic methods, coupled with early clinical symptoms of patients with IA obvious,often delay the best timing of treatment. Recent years, more and more researchers are focusing on the serological testing in order to find specific antigens or antibodies.In this study, based on our previous proteomics, screening A. fumigatus cell wall protein heat shock protein 70(Hsp70) and hydrophobic protein 1(Hyp1) as the goal,to be the recombinant plasmid A. fumigatus Hyp1 and Hsp70 proteins in prokaryotic expression vector E. coli induced for further studying Hsp70 and Hyp1 antigen antibody system in diagnosis of invasive aspergillus infection value and significance lay the foundation.According to A. fumigatus Hsp70 and Hyp1 gene sequences Gen Bank reported, primers were designed to A. fumigatus IFM40808 as a template,PCR amplification of the target gene; by cloning and subcloning gene, the target gene expression vector Pet28a(+) connection; the recombinant plasmid was transformed into E. coli competent cells BL21(DE3), induced by IPTG. Next to purify the targetprotein,and Western-blot analysis of qualitative and quantitative analysis of BCA.Results: 1.The gene of Hsp70 and Hyp1 were obtained successfully by PCR.Sequence analysis showed that the target gene with Gen Bank reported were homologous basically. 2.The recombinant expression plasmid p ET28a(+)- Hsp70 and p ET28a(+)- Hyp1 had been constructed successfully. The target protein were expressed in E. coli after induction with IPTG. 3. The inclusion protein Hsp70 were denaturation and refolding dissolved, in order to obtain a higher purity of the target protein. 4.The results of Western blot suggested that the target protein were capable of specifically reacting with His-tag Ig G. Conclusion: 1. Successfully constructed prokaryotic expression vector p ET28a(+)- Hsp70 and p ET28a(+)- Hyp1. 2. The recombinant expression plasmid p ET28a(+)- Hsp70 and p ET28a(+)- Hyp1 were expressed in E. coli BL21(DE3) successfully, which laid a foundation for the further research in the early diagnosis of invasive aspergillosis.