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Cell strain for expressing HLA-G5 isomer standard protein and application thereof

An HLA-G5, isoform technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., can solve the problems of indistinguishable, difficult to distinguish, lack of standard reference, etc.

Inactive Publication Date: 2020-10-30
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 seven HLA-G isoform molecules have. Detection of 7 HLA-G isoform molecules containing the α1 domain, and 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-G5 isomer standard protein and application thereof
  • Cell strain for expressing HLA-G5 isomer standard protein and application thereof
  • Cell strain for expressing HLA-G5 isomer standard protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: HLA-G1~-G7 isoform gene cloning and pVITRO2-mcs-HLA-G recombinant plasmid construction

[0032] RT-PCR amplification of the HLA-G1~-G7 isoform gene sequence encoding the enzyme cleavage site, using the human choriocarcinoma cell line JEG-3 gene as a template, carry out HLA-G1~-G7 isoform according to the following conditions Amplification of conformational mRNA. The primer sequences and the length of the amplification products are shown in Table 1:

[0033] Table 1 HLA-G1~-G7 RT-PCR amplification primer sequences

[0034]

[0035] Note: The sequences in capital letters are the sequences of endonucleases EcoR I and Xho I.

[0036] PCR reaction system:

[0037] h 2 O 50 μL; Buffer (10×) 5 μL; Mg 2+ (25mmol / L) 2μL; 3′-Primer (25μmol / L) 2μL; 5′-Primer (25μmol / L) 2μL; dNTP (20mmol / L) 1μL; Template (10ng / μL) 1μL; Taq Polymerase (5U / μL) 1 μL.

[0038] PCR program:

[0039] 1. Pre-denaturation at 95°C for 5 minutes

[0040] 2. Denaturation at 94°C for 60...

Embodiment 2

[0046] Example 2: Identification of K562 cell lines stably expressing HLA-G1~-G7 isoforms

[0047] RT-PCR was used to identify the mRNA expression of HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7 isoforms in transfected cells: Trizol reagent was used to extract the total mRNA of each transfected cell line , no degradation was identified by formaldehyde-denaturing agarose gel electrophoresis, A 260 / 280The ratio is 2.0019. 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 4 minutes; 35 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes; and finally 72°C for 10 minutes. Take 5 μl of the PCR product for agarose gel electrophoresis and observe the results. The HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7 specific bands amplified by RT-PCR were in line with the expected target fragment length. The results showed that HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7 isofor...

Embodiment 3

[0050] Example 3: HLA-G isoforms are used as standard proteins in antibody development and screening ( Image 6 )

[0051] Development of new antibody 1 ( Image 6 Panel A in the middle): After HLA-G5 and HLA-G6 standard proteins were electrotransferred to the membrane, they were blocked with 5% non-fat milk powder at room temperature for 4 hours, and washed with 0.2% TBS (Tewen-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 show that: the results show that the new antibody 1 can specifically recognize HLA-G5 and HLA-G6 standard proteins.

[0052] Development of new antibody 2 ( Image 6 Part B in the figure): After HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7 standard proteins were electrotransferred to the membrane, they w...

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Abstract

The invention provides a cell strain for expressing HLA-G5 isomer standard protein, the preservation mechanism is China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202015. The HLA-G5 isomer standard protein can be stably expressed, and can be applied to human leukocyte antigen-G isomer molecule HLA-G5 flow cytometry, immunoblotting, tissue and cell immunohistochemistry, HLA-G isomer function research, specific antibody development and screening as a standard reference substance and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to seven known human leukocyte antigen-G (HLA-G) isomer molecules (HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7 ) expression vector construction and stable expression cell lines, can be used as specific HLA-G isoform molecules (HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7) flow cytometry, Western blotting, tissue and cell immunohistochemistry, HLA-G isoform function research and specific antibody development and screening as standard reference materials and other applications. Background technique [0002] Human leukocyte antigen-G (human leukocyte antigen-G, HLA-G) gene, with a total length of 6.0kb, is located at 6p21.3 on the short arm of human chromosome 6. During protein translation, the first exon of HLA-G mRNA encodes the signal peptide, the second, third and fourth exons encode the α1, α2 and α3 domains of the extracellular region, respectively, and the fifth exon encodes Transme...

Claims

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Application Information

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