Cell strain K562 with specific expression of HLA-G1 antigen

A technology of HLA-G1 and cell lines, applied in the field of tumor cells, to achieve the effect of complete design and simple operation

Inactive Publication Date: 2013-10-02
浙江省台州医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is still a lack of standards for scientific methods such as immunoblotting to distinguish HLA-G isoform molecules based on different molecular weights, and it is necessary to prepare high-purity specific HLA-G isoform molecules as a reference

Method used

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  • Cell strain K562 with specific expression of HLA-G1 antigen
  • Cell strain K562 with specific expression of HLA-G1 antigen
  • Cell strain K562 with specific expression of HLA-G1 antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Cloning of HLA-G1 gene and construction of pVITRO2-mcs-HLA-G1 recombinant plasmid

[0022] PCR amplification of the HLA-G1 gene sequences encoding restriction sites: design the following pair of primer sequences:

[0023] Upstream primers, the underlined part is EcoR I restriction site: 5'-TCGA GAATTC ATGGTG GTCATGGCGCCCCGAA-3'; (SEQ ID NO.1)

[0024] Downstream primers, the underlined part is xho I restriction site: 5'-TCGA CTCGAG TCAATC TGAGCTCTTCTT-3' (SEQ ID NO. 2).

[0025] Using the genome of human choriocarcinoma cell line JEG-3 as a template, HLA-G1 PCR amplification was carried out according to the following conditions, and the fragment size was 1037bp.

[0026] PCR system:

[0027] h 2 O: 50 μL

[0028] Buffer(10×): 5μL

[0029] Mg 2＋(25mmol / L): 2μL

[0030] Primer-up (25μmol / L): 2μL

[0031] Primer-down (25μmol / L): 2μL

[0032] dNTP(20mmol / L): 1μL

[0033] Template (Genome of FRI 100, 10ng / μL):...

Embodiment 2

[0042] Example 2: Identification of K562 cell line stably expressing HLA-G1 antigen

[0043] RT-PCR was used to identify the mRNA expression of HLA-G1 in transfected cells: Trizol reagent was used to extract the total mRNA of each transfected cell line, and no degradation was identified by formaldehyde-denaturing agarose gel electrophoresis, A 260 / 280 The ratio is 2.0019. Take 2 μl of total mRNA and follow the instructions of Fermentas Reverse Transcriptase Kit to synthesize the first strand of cDNA. The parameters of the PCR reaction were: pre-denaturation at 94 °C for 4 min; 35 cycles of 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C; and a final extension at 72 °C for 10 min. Take 5μl of PCR product for agarose gel electrophoresis and observe the results. The specific bands amplified by RT-PCR conform to the expected target fragment length: HLA-G1 (1037bp) (such as Figure 4 shown). The results showed that the HLA-G1 expression-negative K562 cells successfully expr...

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Abstract

The invention discloses a cell strain with specific expression of HLA-G1 antigen. The preservation number is CCTCC: C201351. The preservation date is on May 8, 2013. The preservation place is China Center for Type Culture Collection. The molecular weight of the HLA-G1 antigen produced by the cell strain is 39 kDa. The HLA-G1 antigen comprises heavy chains, a light chain and antigen peptides. The heavy chains comprise Alpha 1, Alpha 2 and Alpha 3 structural domains and transmembrane domains, and intracellular peptide fragments. The light chain is Beta 2 microglobulin (Beta 2 m) with a molecular weight of 12 kDa. The Beta 2 microglobulin is coded by the human 15th chromosome. The antigen is advantaged by strong specificity, good stability and good commercialization application values.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a tumor cell stably expressing membrane-bound HLA-G1. Background technique [0002] In 1987, Geraghty and others successfully cloned the human leukocyte antigen-G (human leukocyte antigen-G, HLA-G) gene for the first time. Alternative splicing of HLA-G initial transcription products produces 7 mature mRNAs, which encode 7 protein isoform molecules, including four membrane-bound HLA-G molecules (HLA-G1, -G2, -G3, -G4 ) and three soluble HLA-G molecules (HLA-G5, -G6, -G7) (see figure 1 ). The molecular weights of HLA-G1~-G7 isomers are 39kD, 31kD, 23kD, 30kD, 37kD, 27kD and 17kD, respectively. Under physiological conditions, HLA-G antigen is only limitedly expressed in a few immune-privileged tissues, such as thymus, cornea, pancreatic islets, and mesenchymal stem cells. However, under pathological conditions, HLA-G molecules can be expressed in a variety of malignant tumor microen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
Inventor 颜卫华林爱芬许惠惠徐丹萍
Owner 浙江省台州医院
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