A nanobody against human parathyroid hormone and its screening method and application
A parathyroid hormone and nano-antibody technology, applied in the fields of biochemistry and molecular biology, can solve the problems of increasing activity changes, destroying protein activity, and not being able to guarantee the binding ability of PTH, so as to achieve the effect of easy deformation
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0051] Example 1 Construction and Identification of Recombinant Plasmid pET-23a-PTH-C
[0052] (1) Obtaining the target gene
[0053] The target protein with sulfhydryl tag was obtained by PCR method. Using the plasmid pET-23a-PTH (preserved by the Biomaterials Research Group of the School of Life Science and Technology, Dalian University of Technology) as a template, rationally design primers on this basis, and introduce a cysteine codon at the end of the PTH gene. The points are Nde I and Bam HI.
[0054] Upstream primer (UP):
[0055] 5'-CTACACGGAAATTCCATATGTCTGTTTCTGAAATCCAGC-3'
[0056] Downstream primer (DOWN):
[0057] 5'-GACTATGTCCGGGATCCTTAGCACTGAGATTTAGCTTTGGTCAGAACG-3'
[0058] (2) Purification of PCR products
[0059] After the reaction, agarose gel (1%) electrophoresis was performed, and the electrophoresis results showed that the amplified gene fragment presented a bright band around 270bp. The gel recovery kit (TaKaRa Code: 9762) produced by TaKaRa Comp...
Embodiment 2
[0067] Embodiment 2 Shake flask culture and expression of fusion protein PTH-C
[0068] According to the method of Example 1, the constructed plasmid pET-23a-PTH-C was transferred into the expression host E.coli BL21(DE3), and after overnight culture, positive colonies grew on the culture dish. Pick 4 single colonies and inoculate them into 20mL Amp-LB liquid medium respectively, and activate overnight at 170r / min at 37°C.
[0069] Inoculate 1% of the activated strain into LB medium, and after culturing for 3-4 hours, add IPTG with a final concentration of 0.2mM for induction, stop the induction after 4 hours, and collect the bacteria by centrifugation. SDS-PAGE was carried out on the whole bacteria, and the results were as follows: Figure 16 As shown, the lane M is the protein low-component marker, the lane 1 is the whole bacterial protein before IPTG induction, and the lanes 2-5 are the four positive bacteria of the randomly selected strains (named in turn No. 1-4 strains) i...
Embodiment 3
[0070] Example 3 Separation and purification of fusion protein PTH-C and detection of sulfhydryl groups
[0071] (1) Solubility investigation of recombinant protein
[0072] First, the cultivated No. 4 bacteria were broken up by ultrasonic breaker, centrifuged at 4°C and 10,000rpm, and the supernatant and precipitate were taken respectively for SDS-polyacrylamide gel electrophoresis. Most of the expressed target protein (9600Da) was distributed in In the supernatant, soluble expression was present.
[0073] (2) Protein pretreatment - coarse separation
[0074] PEI with a final concentration of 0.12% (w / v) was added to the supernatant obtained by crushing and centrifugation to remove nucleic acids in the solution. Stand at 4°C for 30min, centrifuge at 10000r / min for 15min at 4°C. Take out the supernatant, add an equal volume of saturated ammonium sulfate, let stand at 4°C for 30 min, and centrifuge. Retain the precipitate and dissolve it with pH 3.5 citrate buffer, stir wel...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More