Preparation of extramembranou section of therapeutic protein B lymphocyte stimulation factor
A technology of B lymphocytes and stimulating factors, which is applied in the fields of peptide/protein components, medical preparations containing active ingredients, genetic engineering, etc., can solve the problems that the heat transfer rate cannot meet the technological requirements and the production cost is high
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0063] Cloning of BlyS Coding Region (ORF) and BlyS Extramembrane BlyS-C Sequence
[0064] In order to obtain the ORF sequence of BlyS, the following pair of primers were designed according to the BlyS nucleotide sequence:
[0065] BlyS-F3: 5'-ATCGGAATCCTCACGCCTTACTTCTTGCC-3' (SEQ ID NO: 5)
[0066] BlyS-R3: 5'-ATCGAAGCTTCATGGTGTAAGTAGGTCACAGC-3' (SEQ ID NO: 6)
[0067] The above primers introduced EcoR I and Hind III restriction sites respectively, and the amplified products covered the corresponding DNA sequence from the 10th to the 288th amino acid of BlyS. Using BlyS-F3 and BlyS-R3 as primers, a fragment of about 850 bp was amplified from a human fetal brain library (Clontech). The PCR amplification conditions were: 94°C for 50 sec, 53°C for 1 min, 72°C for 2 min, and the last cycle of extension for 9 min. The PCR product was purified and identified by digestion with Pst I and Dra I respectively, and it was preliminarily confirmed that it was the ORF sequence of BlyS. ...
Embodiment 2
[0073] Construction of expression vector pT7450-BlyS2
[0074] Plasmid pT7-ZZa (Novagen Inc.) was digested with EcoRI and NcoI, and the oligonucleotide fragment 5'-CATGGCATATGG-3' (SEQ ID NO: 9) and 3'-CGTATACCTTAA-5' (SEQ ID NO: 10) was used to ligate the linker formed by T4 DNA ligase, and transform Escherichia coli DH-5α. After digestion and screening, the recombinant plasmid pT7450 was obtained. Plasmid pGEM-BlyS2 was digested with Nde I and Hind III, and the BlyS2 fragment was purified by agarose gel electrophoresis and ligated with the pT7450 vector digested with Nde I and Hind III. Escherichia coli DH-5α was transformed. After enzyme digestion and screening, the recombinant plasmid pT7450-BlyS2 ( figure 1 ) into Escherichia coli JM105. Sequencing proved that the sequence of the recombinant plasmid pT7450-BlyS2 was spliced correctly.
Embodiment 3
[0076] Expression of BlyS-C gene
[0077] The plasmid pT7450-BlyS2 obtained in Example 2 was used to transform Escherichia coli BL21(DE3), and the transformants were inoculated in LB liquid medium containing ampicillin, and cultured with shaking at 37°C until A600=0.5. Add IPTG to a final concentration of 1 mmol / L, and continue shaking culture at 37°C for 4 hours. Bacterial samples were taken before and after IPTG induction and detected by SDS-PAGE electrophoresis. The results showed that about 18KD BlyS-C protein was expressed after induction with IPTG.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More