The cDNA cloning of human granulysin Mr 15000 and Mr 9000 active segments from CTL activated by allogenic antigen / 重庆医科大学学报

Objective; To clone, sequence and analyze the coding sequences of the Mr 15000 and Mr 9000 active segments of natural granulysin derived from human CTLs activated by allogenic antigen ,in order to establish the basis for further purifying and investigating the immune - impairing mechanism of granulysin. Methods; The coding sequences of the Mr 15000 and Mr 9000 granulysin gene were amplified from the total RNA of activated CTLs of healthy person after reverse transcription by Nested - PCR, inserted into pET32a ( + ) vectors and then transformed into E. Coli TOP10, respectively. The recons were identified by PCR, endonuclease digestion and sequencing. Results: The whole coding sequences of the Mr 15000 and Mr 9000 active segments were successfully cloned as expected. The accurate pET32a - Mr 15000 and pET32a - Mr 9000 recons were obtained through Colony - PCR, endonuclease digestion and sequencing. There lied polymorphism on the 119 th amino acid of the product encoded by GLS gene. Conclusion; The coding sequences of the Mr 15000 and Mr 9000 active segments of human granulysin can be obtained and cloned by the methods mentioned above and can be used for subsequent research.

Detection of Adenovirus and Parvovirus in CSF Specimens from Viral Meningitis Adult Patients by Enzyme Immunoassay with Monoclonal Antibody and by Nested - PCR

Viral meningitis and encephalitis are important and serious diseases in young children and adults. There are many causative viruses but it is known that a low percentage of adenovirus (ADV) and parvovirus (PA V) infected individuals develop meningitis or encephalitis. Few reports have been published about central nervous system complications that were rare but fatal. First we used enzyme immunoassay (EIA) with monoclonal antibody to detect ADV antigen (Ag) and PAV Ag in cerebrospinal fluids (CSF) from acute phase of hospitalized adult patients with viral meningitis or viral encephalitis. Second we detected ADV DNA and PAV DNA in the same CSF after cell culture by nested-polymerase chain reaction (PCR). Third we evaluated ADV and PAV dual infection in CSF by EIA and nested-PCR. ADV Ag in CSF by EIA positivity was 42.9% (12l28) and PAV Ag in CSF by EIA positivity was 21.4% (6/28). ADV DNA in CSF by nested-PCR positivity was 89.3% (25/28) and PAV DNA in CSF by nested-PCR positivity was 38.5% (10/26). ADV and PAV dual infection in CSF by 11CSted-PCR was 35.7% (10/28). Detection rate of ADV DNA and PAV DNA in CSF by nested-PCR with viral meningitis or encephalitis adult patients were higher than we expected. Positive detection of nested-PCR was higher than that of EIA with monoclonal antibody for detection of antigens ADV and PAV in CSF with viral meningitis or encephalitis adult patients. Both methods were analnized by the McNemar test.