Novel serine protease BSSP2
a serine protease and serine protease technology, applied in the field of serine protease isolated polynucleotides of human and mouse serine proteases, can solve the problems of severe disability in daily life or social life, substantial inconsistency between clinical diagnosis and autopsy diagnosis, and the definition of alzheimer's disease based on lowering the blood flow from parietal lobe to temporal lobe is very dangerous
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example 1
Cloning of Novel Serine Protease mBSSP2 Gene
[0103] The cloning was carried out by PCR using a mouse brain cDNA library (Clontech) as a template and nucleotide sequences corresponding to an amino acid sequence common to serine proteases represented by
Primer 1:GTG CTC ACN GCN GCB CAY TG(SEQ ID NO: 20)Primer 2:CCV CTR WSD CCN CCN GGC GA(SEQ ID NO: 21)
[0104] as primers. Namely, 5 μl of the template, 5 μl of 10×ExTaq buffer, 5 μl of dNTP, 10 pmol of each of the above primers and 0.5 μl of ExTaq (TAKARA) were added and the total volume was adjusted to 50 μl with sterilized water. PCR was carried out by repeating a cycle of heating at 94° C. for 0.5 minute, at 55° C. for 0.5 minute and then at 72° C. for 1 minute, 30 times. The PCR product was mixed with pCR II-TOPO vector attached to TOPO TA cloning kit (Invitrogen) and the mixture was allowed to stand at room temperature for 5 minutes. Then, according to a conventional manner, E. coli Top 10 attached to the kit was transformed and app...
example 2
Expression mBSSP2 Gene in Mice Internal Organs
[0105] According to the protocol of QuickPrep Micro mRNA purification Kit (Amersham-Pharmacia), mRNAs were isolated from various internal organs of Balb / c mice or their fetuses. They were subjected to electrophoresis according to a conventional manner and transcribed to a nylon membrane. A probe was prepared separately by isolating a part of a nucleotide sequence encoding the mature protein of mBSSP2 from pCR II / mBSSP2, purifying it and labeling it with α-32P dCTP. The probe was diluted with 5×SSC and reacted with the above membrane filter at 65° C. for a whole day and night. Then, the filter was washed twice each with 2× / 0.1% SDS at room temperature for 30 minutes, 1× / 0.1% SDS at room temperature for 30 minutes and 0.1× / 0.1% SDS at 65° C. for 30 minutes. The filter was exposed to an imaging plate for FLA2000 (Fuji Film) for one day to analyze the expression. The results shown in the drawings are those obtained by using mRNAs prepared f...
example 3
Expression of Novel Serine Protease Mature Protein Encoded by mBSSP2 Gene
(1) Construction of Expression Plasmid
[0107] A cDNA region encoding the mature protein of BSSP2 protein was amplified by PCR using the plasmid pCR II / mBSSP2 as a template (the sequence of the 1st to 717th bases of SEQ ID NO: 1 was amplified by using the primers having the sequences represented by SEQ ID NOS: 25 and 29). The PCR product was ligated to pTrc-HisB (Invitrogen) which had been digested with BamHI and blunted with mung bean nuclease. E. coli JM109 was transformed by the resultant and colonies formed were analyzed by PCR to obtain E. coli containing the desired serine protease expressing plasmid pTrcHis / mBBSP2.
[0108] The resultant E. coli was designated E. coli pTrcHis / mBSSP2 and deposited at National Institute of Bioscience and Human-Technology (NIBH), Agency of Industrial Science & Technology of 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan on Oct.29, 1998 under the accession numbers of FERM P-1...
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