Analysis Of The DHFR And DHPS Genes Of Pneumocystis Carinii Infected SD Rats

Objective:To analyse the dihydrofolate reductase(DHFR) and dihydropteroate synthase (DHPS) genes sequence features of pneumocystis carinii infected SD rats.To analyse the DHPS gene mutation applied PCR-linked restriction fragment length polymorphism (PCR-RFLP) technique.Methods:(1) SD rats model of Pneumocystis carinii(PCP) were established by groin subcutaneous injection with dexamethasone sodium phosphate.(2) The PCP model rats were randomly divided into sulfanilamide treatment group and positive control group,the normal rats were randomly divided into normal control group and negative control group.(3) Pneumocystis carinii(Pc) cysts were observed in the lung print smear after staining with gormori methenamine silver(GMS),and pathological changes in the lung section were observed after staining with hematoxylin-eosin(HE).(4) Collected rats lung tissue,extracted DNA,Pc DHFR and DHPS genes were amplified by PCR method.After purification of PCR products the sequencing was determined.The gene library was searched for homology analysis.(5)The PCR amplification products were digested with Accâ… and Haeâ…¢,and the DHPS gene was analysed by PCR-RFLP technique.Results:(1) The rats of the sulfanilamide treatment group and positive control group were all infected by Pc,the rats of normal control group and negative control group were all not.(2) The Pc DHFR and DHPS genes of the sulfanilamide treatment group and positive control group were all amplified,none of the other groups.The DHFR gene of the sulfanilamide treatment group and positive control group were amplified 832bp.Compared the DHFR gene with the sequence gb[M26495](Pc f.sp.rat),the homology rates were both 99%.Compared it with gb[AF322061](Pc f.sp.rat),the homology rates were both 100%. The DHPS gene of sulfanilamide treatment group was amplified 816bp.Compared the DHPS gene with the sequences gb[U66283](Pc f.sp.muris) and gb[U66282](Pc f.sp.hominis),homology rates were 94%and 83%respectively.The DHPS gene of positive control group was amplified 839bp,compare it with gb[U66283]and gb[U66282],the homology rates were 95%and 84%respectively.(3) The Pc DHPS gene of the sulfanilamide treatment group and positive control group were not found Ala55/Ser57 mutation by PCR-RFLP technique.Conclusion:(1) It is a good way to establish the SD rat-PCP model by groin subcutaneous injection with dexamethasone sodium phosphate.(2) The Pc DHFR and DHPS genes of the sulfanilamide treatment group and positive control group are all amplified.They are highly identical to those of Pc f.sp.rat in Gene bank.(3) The technique of PCR is sensitivity,high resolution,repeatability,simple and rapid.It is applicable to diagnosis the Pneumocystis carinii pneumonia.(4) The establishment of the Pc DHPS genes of the SD rats are wide-type.