Neurotrophic factor NNT-1
A technology of NNT-1 and sympathetic nerves, applied in nervous system diseases, growth factors/inducible factors, anti-growth factor immunoglobulins, etc., can solve problems that few people understand
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Embodiment I
[0232] Embodiment 1: Cloning of cDNA and genome cloning against NNT-1
[0233] A. Construction of cDNA library
[0234] At 37°C, 5% CO 2 In the environment, human T-cell lymphoma cells (Jurkat cells) were grown in RPMI 400 medium containing 10% fetal bovine serum. The medium was buffered (pH 7.5) with 10 mM HEPES. Cells were divided into two groups after 8 passages. One group was grown to confluence (2×10 7 cells / flask) and RNA harvested from these cells was used as "driver" RNA. The other group was the "test substance" group and was activated with the following treatments.
[0235] Cells were activated for 8 hours by adding superantigen streptococcal enterotoxins B and F (TSST) 80 ng / ml, PKC activator PMA 50 ng / ml, calcium ionophore A21832 125 ng / ml. The protein translation inhibitor cycloheximide was also added at a concentration of 1 mg / ml. RNA was harvested from different groups of cells at different time points.
[0236] 1. Preparation of total RNA:
[0237] Ce...
Embodiment II
[0256] Embodiment II: the preparation of recombinant mammalian NNT-1 protein
[0257] An expression vector containing human NNT-1 cDNA and flag-tag peptide was constructed by PCR amplification of the fusion gene. Sense primer with a Hind III site at the 5' end:
[0258] (5'-AGCAAGCTTCACCATGGACCTCCGAGCAGGGGACTC-3')
[0259] (SEQ ID NO: 6)
[0260] It encodes amino acid -27 (Met) to amino acid -21 (Asp), and an antisense primer with a NotI site at the 5' end encodes a flag-tagged peptide and the last 8 amino acids at the 3' end
[0261] (5'AGCGGGGCCGCACTACTTGRCATCGTCGRCGTCCTTGTACTCGAAGCCATGAGCCCCCAGGTGCAG-3') (SEQ ID NO: 7), which were used in PCR to amplify the fusion gene. This fusion gene was ligated to the P CEP4 vector (Invitrogen Inc., San Diego, CA). The expression vector was transfected into EBNA-1-293 cells with lipofection reagent (BRL, Gaithersburg, MD) using the manufacturer's suggested method. 48 hours after transfection, 293 cells and conditioned media were ...
Embodiment III
[0262] Embodiment III: the preparation of recombinant escherichia coli NNT-1 protein
[0263] A cDNA clone of NNT-1 encoding SEQ ID NO: 2 amino acids Leu(1) to Phe(198) was inserted into vector pAMG21, which is a derivative of pCFM 1656 (ATCC Accession No. 69576) and contains the vector for insertion from lux PR Suitable restriction sites for genes downstream of the promoter (see US Pat. No. 5,169,318 for a description of the lux expression system). The host cell used was E. coli K12, strain CGSC6159 (genetic stock of Yale University, New Haven, CT). The host cells were transformed with the vector using standard transformation methods, and then they were cultured at 30°C in 2XYT medium containing about 50 µl / ml kanamycin. Induction of the NNT-1 gene product was initiated by adding the autologous inducer N-(3-oxohexanoyl)-DL-homoserine lactone to the medium to a final concentration of approximately 30 ng / ml, and the culture was grown at Incubate at 30°C or 37°C for approxim...
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