Cell strain for expressing HLA-G1 isomer standard protein and application thereof

A technology of HLA-G1 and isomers, applied in the field of bioengineering, 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-G1 isomer standard protein and application thereof
  • Cell strain for expressing HLA-G1 isomer standard protein and application thereof
  • Cell strain for expressing HLA-G1 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-G1 isomer standard protein, the preservation mechanism is China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202004. The HLA-G1 isomer standard protein can be stably expressed, and the HLA-G1 isomer standard protein can be applied to human leukocyte antigen-G isomer molecule HLA-G1 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): C12N5/10C12N15/85G01N33/96C12R1/91
CPCC07K14/70539C12N15/85G01N33/96G01N2333/70539
Inventor 颜卫华林爱芬许惠惠
Owner TAIZHOU ENZE MEDICAL CENT GROUP
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