PD-L1 immunohistochemical reference substance and preparation method and application thereof
An immunohistochemistry, PD-L1 technology, applied in the field of immunology, can solve the problems of irreproducibility, tissue heterogeneity, unable to provide a stable control of PD-L1 expression intensity, etc., to ensure stability and good uniformity. Sexuality and consistency, the effect of overcoming limitations
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[0044] The present invention also provides a method for preparing the PD-L1 immunohistochemical reference product, comprising the following steps:
[0045] (1) Knock the cDNA sequence of PD-L1 into the cell line by means of homologous recombination to obtain a monoclonal cell line;
[0046] (2) Determine the PD-L1 expression intensity of the monoclonal cell line by qRT-PCR and immunohistochemical staining;
[0047] (3) Arrange any one or more of monoclonal cell line sections with no, weak, moderate and strong staining in PD-L1 immunohistochemistry on a slide, leaving an area for the sample to be tested, As a reference for PD-L1 immunohistochemical staining.
Embodiment 1
[0048] Example 1 Integrating a plasmid containing the PD-L1 gene into a specific site in the cell genome to obtain a stably transformed single-cell clone
[0049] In a transgenic way, preferably, the CRISPR / Cas9 system is used to integrate the plasmid containing the PD-L1 gene into a specific site of the cell genome through homologous recombination. Among them, the plasmid used for integration includes the homology arm, the cDNA sequence of the PD-L1 gene (NM_014143.4) and the resistance screening gene. The integration site can be selected according to requirements, and the preferred site is AAVS1. Specific steps are as follows:
[0050] 1) For the integration site, design a specific guide RNA (guide RNA, gRNA), and construct the expression vector of the guide RNA. The sequence of the guide RNA is: 5'-ggggccactaggcaggat-3'.
[0051] 2) Construct the integrated plasmid pEF1-PDL1-UC57, including homologous left arm, cDNA sequence of PD-L1 gene, resistance screening gene and h...
Embodiment 2
[0059] Example 2 Preparation of Paraffin Embedded Paraffin Blocks for Cells
[0060] Specific steps are as follows:
[0061] 1) The monoclonal cells in Example 1 were cultured in FBS medium, and the cells were collected by centrifugation after the culture.
[0062] 2) Fix the cells with 10% neutral formalin for 48 hours. After the cells were fixed, the cells were resuspended with 70% (v / v) ethanol, and the residual formalin solution was washed away.
[0063] 3) Mix the above-mentioned fixed cell suspension with an equal volume of agarose gel solution, and fix it into shape.
[0064] 4) Use a Leica automatic dehydrator for dehydration.
[0065] 5) After dehydration, the cell mass was put into an embedding mold, and was embedded in paraffin using a Leica HistoCore Arcadia H + C embedding machine. The embedded wax blocks were placed on a Leica RM2255 paraffin microtome for sectioning with a thickness of 3-10 μm.
[0066] 6) Spread the slices on the surface of water at 37°C f...
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