Preparation and application of specific antibody of T cell surface marker molecule CD4-1 of lateolabrax japonicus
A technology of perch and cell subgroups, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, application, specific peptide, etc., can solve the problem of lack of specific antibodies for T lymphoid marker molecules
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0038] Example 1: Gene cloning of T cell surface marker molecule CD4-1 in Chinese sea bass
[0039] 1. Design degenerate primers for the conserved sequence part of the gene
[0040] Find the CD4-1 protein sequences of other fishes that have been published on NCBI, use Blast to find the sequences of fish with high homology to Perciformes, and use BioEdit to find the relatively conserved regions on the sequence (the consecutive identical amino acids are greater than or equal to 6), respectively designed their own degenerate primers (Table 1) according to the conserved regions, and optimized the degenerate primers according to the codon preference of Chinese perch, and sent them to Qingke Company for synthesis.
[0041] Table 1: List of degenerate primers for CD4-1 gene
[0042] F: LISGQQ 5'TTRATTTCDGGCCARCAG 3' R: ITKLIE 5' YTCRATCTTCAGCGTKAT 3'
[0043] 2. Gene core fragment amplification
[0044] Using the designed degenerate primers, the CD4-1 gene w...
Embodiment 2
[0055] Example 2: Preparation of polyclonal specific antibody against Chinese perch CD4-1
[0056] 1. Screening of CD4-1 antigenic peptide fragments
[0057] The protein secondary structure domain of the Chinese sea bass CD4-1 molecule obtained in Example 1 was analyzed. The results showed that the transmembrane region of Chinese perch CD4-1 molecule is located in the 419-441 region, of which the 1-418 region is the extracellular region, and there are four Ig-like domains in the extracellular region, and the 442-470 is the intracellular region. Area. The purpose of predicting the transmembrane region and structural domain according to SMART software is to ensure that the antigenic peptide designed for analysis is located on the extracellular region and Ig-like domain of Chinese sea bass CD4-1. Using the online prediction system Swiss model of protein tertiary structure, https: / / swissmodel.expasy.org / interactive , conducted homologous construction and analysis on the tertia...
Embodiment 3
[0067] Embodiment 3: the immunoblotting method identification of the prepared polyclonal gene of the present invention
[0068] (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis:
[0069] ① Add the purified Chinese perch CD4-1 recombinant protein and Chinese perch kidney leukocytes to the sample buffer containing sodium dodecylsulfonate in equal proportions, and boil in boiling water for 3-5 minutes;
[0070] ②Put the sample treated in ① into the sample well, add 10 μl of sample into each well, under constant current condition, use low current (30-40mA) at the beginning, after the sample is concentrated into a line in the stacking gel, Increase the current (50-70mA) and electrophoresis until the bromophenol blue indicator reaches the bottom edge, then stop the electrophoresis and take out the gel; part of the gel is used for membrane transfer, and part is stained in Coomassie brilliant blue staining solution for 1 hour.
[0071] ③Cut a piece of PVDF membrane with ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More