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Cell strain for expressing HLA-G isomer standard protein with deletion of alpha1alpha2 structural domain and use thereof

A technology for HLA-G and isoforms, which is applied to cell lines expressing HLA-G isoform standard proteins with α1α2 domain deletion and its application field, which can solve the problems of indistinguishability, lack of standard reference, and difficulty in distinguishing

Inactive Publication Date: 2021-08-20
TAIZHOU ENZE MEDICAL CENT GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been widely used at present. The antibody 4H84 expressed by HLA-G molecules is detected by immunohistochemistry or Western blotting. Its recognition site is located in the α1 domain of the extracellular region that all 7 HLA-G isoform molecules have. In terms of specificity, it can Detect 7 HLA-G isoform molecules containing the α1 domain, but the expression of specific HLA-G isoform molecules cannot be distinguished in immunohistochemistry
At the same time, the molecular weights of HLA-G1~-G7 isomers are 39kD, 31kD, 23kD, 30kD, 37kD, 27kD and 16kD, respectively. Due to the lack of specific HLA - Standard reference for G isoform molecules, it is not easy to distinguish the expression of specific HLA-G isoform molecules in Western blot detection

Method used

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  • Cell strain for expressing HLA-G isomer standard protein with deletion of alpha1alpha2 structural domain and use thereof
  • Cell strain for expressing HLA-G isomer standard protein with deletion of alpha1alpha2 structural domain and use thereof
  • Cell strain for expressing HLA-G isomer standard protein with deletion of alpha1alpha2 structural domain and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: HLA-G-△α1 and HLA-G-△α1α2 isoform gene cloning and pVITRO2-mcs-HLA-G recombinant plasmid construction

[0023] Using HLA-G5 mRNA as the amplification template, the amplification primers for the gene encoding HLA-G-△α1 are as follows:

[0024] Upstream primer: 5'-TCGA GAATTC ATGAGTTCTCACACCCTCAGT-3', the underlined part is the EcoRI restriction site, and the upstream primer is the sequence of Seq No.5 in the sequence list.

[0025] Downstream primer: 5'-TCGA CTCGAG CCACCGACCCTGTTA-3', the underlined part is the XhoI restriction site, and the downstream primer is the sequence of Seq No.6 in the sequence list.

[0026] The HLA-G-△α1 isoform mRNA was amplified according to the following conditions, and the fragment size was 620bp.

[0027] Using HLA-G5 mRNA as the amplification template, the amplification primers for the gene encoding HLA-G-△α1α2 are as follows:

[0028] Upstream primer: 5'-TCGAGAATTCATGGACCCCCCCAAGACACACGTGA-3', the underlined part is the ...

Embodiment 2

[0044] Example 2: Identification of K562 cell lines stably expressing HLA-G-△α1 and HLA-G-△α1α2 isoforms

[0045] RT-PCR was used to identify the mRNA expression of HLA-G-△α1 and HLA-G-△α1α2 isoforms in transfected cells: Trizol reagent was used to extract the total mRNA of each transfected cell line, and it was electrophoresed on formaldehyde denaturing agarose gel No degradation was identified, A 260 / 280 The ratio was 2.06. Take 2 μl of total mRNA and reverse transcribe to synthesize the first strand of cDNA. The PCR reaction parameters were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes, a total of 35 cycles; finally, extension at 72°C for 10 minutes. Take 5 μl of the PCR product for agarose gel electrophoresis and observe the results. Such as image 3 As shown, the specific bands amplified by RT-PCR conformed to the expected target fragment lengths: HLA-G-△α1 (633bp) and HL...

Embodiment 3

[0049] Example 3: HLA-G-△α1 and HLA-G-△α1α2 as standard proteins in antibody development and screening

[0050] As a standard protein in new antibody screening: HLA-G-△α1 (preservation number: CCTCC NO: C202046) and HLA-G-△α1α2 (preservation number: CCTCC NO: C202047) were lysed to obtain protein electrotransfer membranes, and then used 5 Block with % skimmed milk powder at room temperature for 4 hours, and wash with 0.2% TBS (Teween-20 PBS). Add new antibody 1, detect its recognition specificity, incubate overnight at 4°C, and wash; add HRP-labeled rabbit anti-mouse IgG antibody, incubate at room temperature for 30 minutes, wash with Dako REAL TM EnVision TM The detection system (DAKO) was incubated for 1-3 minutes. The results showed that: part of the new antibody cell culture supernatant (such as Figure 5 #18 cell line supernatant) can specifically recognize HLA-G-△α1 standard protein. Other cell culture supernatants are non-specific recognition, which provides a basis...

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Abstract

The invention provides a cell strain for expressing an HLA-G isomer standard protein with deletion of an alpha1alpha2 structural domain and use thereof. The cell strain is deposited in China Center for Type Culture Collection and has the deposit number of CCTCC NO:C202047. The cell strain can express an HLA-G isomer standard protein with deletion of an alpha1alpha2 structural domain, and can be used as a standard reference substance for flow cytometry, immunoblotting, tissue and cell immunohistochemistry, HLA-G isomer function research and specific antibody development and screening.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to the construction and stable expression of the novel HLA-G isomer HLA-G-Δα1 and HLA-G-Δα1α2 expression vectors with deletion of the extracellular α1 domain and the deletion of the α1α2 domain. As a specific HLA-G isoform molecule HLA-G-Δα1 and HLA-G-Δα1α2 flow cytometry, Western blot, tissue and cell immunohistochemistry, HLA-G isoform functional research and specific antibody development and screening Applications such as standard reference objects. Background technique [0002] Human leukocyte antigen-G (human leukocyte antigen-G, HLA-G) gene was discovered and cloned by Geraghty in 1987. HLA-G molecules were first discovered in 1990 in extravillous trophoblast cells at the maternal-fetal interface, and the expression of HLA-G was first observed in melanoma tumor tissues and cells in 1998. Functional studies have shown that HLA-G molecules can regulate the biological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/12C07K14/74G01N33/68C12R1/91
CPCC12N5/0694C12N15/85C07K14/70539G01N33/6854C12N2510/02C12N2800/107G01N2333/70539
Inventor 颜卫华林爱芬张盈盈
Owner TAIZHOU ENZE MEDICAL CENT GROUP
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