Prokaryotic Expression Of P30 Adhesin Gene Of Mycoplasma Pneumoniae And Preliminary Application Of The RP30 Protein

PART I Prokaryotic expession of P30 adhesin gene ofMycoplasma pneumoniae and preliminary analysis of the rP30proteinObjective: To construct the prokaryotic expression vector carrying intactP30 gene and express P30 in E.coli, and to purify the rP30 proein withaffinity chromatography technique. Then the potential value of rP30protein in immunodiagnosis for MP infections was evaluated by clinicalassay.Methods: The only one "UGA" code in P30 gene was changed into"UGG" by modifying the primer. The P30 gene was amplified by PCRfrom MP genome, then cloned into pET32a(+) plasmid. The recombinantplasmid pET32a(+)/P30 was transformed into E.coli BL21. With 1mMIPTG induction the rP30 was expressed. The rP30 was identified bySDS-PAGE and purified with Ni-NTA column. The antigenicity of rP30was confirmed by Western-blot. An indrect dolt-ELISA based on the rP30was preliminary applied to detect the IgG in sera from 40 patients whowere suspected to MP infection.Result: The PCR products without "UGA" code was obtained,and thesequence was consistented with the P30 gene in Genbank.The plasmidpET32a(+)/P30 was constructed successfully. A recombinant protein about52kD was expressed in BL21(DE3). SDS-PAGE showed that therecombinant protein mainly expressed as inclusion bodies form. The rP30was purified through Ni-NTA resin and the purity of target protein was93%. Western-blot suggested that the rP30 could be recognized by serafrom the patients infected with MP. The specificity and sensitivity of theinderected dolt-ELISA based on rP30 was 72.22% and 95.45%.Conclusion: The prokaryotic expression vecter pET32a(+)/P30 wasconstructed successfully, and the rP30 was obtained. This provide anexperimental basis for further study of the pathogenesis and serologicaldiagnosis for MP infection. PART II Establishment of a mouse pneumonia model forMycoplasma pneumoniae infectionObjective: To establish a mouse pneumonia model for MP infection andquest for the courses of pneumonia in mouse.Methods: BALB/C mice were randomly grouped. Group I wasinoculated intranasally with MP suspension on day 0,1,2, group II wasdone on day 0,8,9, and the control groups were set up with sterile normalsaline. Mice were sacrificed on 3~18 days. MP was detected by culturingthe lung tissues and BALF of each mouse. Lung tissue sections wereexamined and a histopathological scoring system was applied to juge theseverity of the inflammatory.Result: All of the mice were survived after inoculated. The infectedanimals showed inflammatory changes in lung with vary grades. Thecultures of MP in lung and BALF speciments of infected animals werepositive, and negative in control groups. Histopathologic scores rangedfrom 1.5 to 14.3.Conclusion: The mouse pneumonia model for MP infection wasestablished successfully, and the histopathological scoring system can beused to evaluate the severity of acute inflammatory lesions of lung onBALB/C mice. The model provide an experimental basis for further studyof the pathogenesis and immunological state after MP infected.