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Mouse DCF1 specific polyclonal antibody and preparation method of mouse DCF1 specific polyclonal antibody

A polyclonal antibody and specific technology, applied in the field of mouse DCF1 specific polyclonal antibody and its preparation, can solve the problems of poor recognition effect and the like

Inactive Publication Date: 2012-10-03
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] According to the information available so far, the only commercially available relevant antibodies for DCF1 are anti-human polyclonal antibodies. Although the instructions indicate that they can also resist mouse-derived DCF1, this recognition effect is found to be very poor in practical applications; There is no commercial antibody against mouse DCF1

Method used

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  • Mouse DCF1 specific polyclonal antibody and preparation method of mouse DCF1 specific polyclonal antibody
  • Mouse DCF1 specific polyclonal antibody and preparation method of mouse DCF1 specific polyclonal antibody
  • Mouse DCF1 specific polyclonal antibody and preparation method of mouse DCF1 specific polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction and identification of pGEX-4T1-59a and pET-30a-59c prokaryotic expression vectors

[0027] Using TMHMM Server v.2.0 to predict the protein transmembrane structure of DCF1 amino acid sequence, using Premier 5.0 software and the principle of no frameshift mutation in the expressed fusion protein to design the upstream of DCF1 extramembrane antigen sequence and Elisa substrate protein sequence and downstream primers, introducing B in its upstream and downstream primers, respectively wxya I and S al The enzyme cutting site of I and the corresponding protective bases, and the start codon and stop codon were added to the upstream and downstream primers respectively, and the primers were synthesized by Shanghai Yingwei Jieji Company. The upstream primer of the antigen sequence is: 5'-CGC GGATCC atgGACACAGCGTCCTGTCAC-3', the downstream primer is: 5'-CAAGAC GTC GAC ctaCAGCCTGCAGCCCTCTCTGG-3'; the upstream primer of the protein sequence for the Eli...

Embodiment 2

[0028] Example 2: Soluble Expression and Purification of Antigen Polypeptide Fusion Protein

[0029]Heat-shock the pGEX-4T1-59a vector into BL21(DE3) competent medium. After colonies grow on the plate, use a sterilized toothpick to pick a single colony into a test tube containing 5mL LB medium, shake at 37°C and 220rpm Cultivate overnight, add 1% of the inoculum, that is, 2mL of bacterial solution, into a conical flask containing 200mL of LB medium, and shake the OD at 37°C 600 When it was 0.8, IPTG was added to a final concentration of 0.5mM for induction culture, and after shaking culture at 37°C for 4h, centrifuge at 6000rpm for 5min to collect the bacteria into a 50 mL centrifuge tube. Place the 50 mL centrifuge tube with collected cells on ice, add 10 mL of pre-cooled cell sonication buffer, 300W, 30s sonication, 15s pause, and last for 30min. After complete crushing, put the centrifuge tube into a centrifuge, centrifuge at 11,000 rpm for 15 minutes at 4°C, collect the...

Embodiment 3

[0031] Example 3: Expression and purification of Elisa substrate protein

[0032] Heat-shock transformation of the pET-30a-59c vector into BL21(DE3) competent cells, subsequent expanded culture, induced fermentation and sonication were the same as in Example 2. Take a small amount of supernatant and precipitate respectively as samples for SDS-PAGE fusion protein soluble expression analysis, see Figure 5 -A. The results showed that the target protein was present in the supernatant phase.

[0033] Use the His tag to purify the target protein in the supernatant. For the specific process, refer to the Ni Sepharose 6 Fast Flow product manual of Amersham Biosciences, and take the components in the purification process for SDS-PAGE analysis, see Figure 5 -B. The results showed that the target protein was mainly present in the eluate, the purification was successful, and it could be used as the substrate coating protein for subsequent Elisa detection.

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Abstract

The invention relates to a mouse DCF1 specific polyclonal antibody and a preparation method of the of mouse DCF1 specific polyclonal antibody. The specific polyclonal antibody is obtained by binding a polypeptide specificities of 44th-76th amino acid residues in SEQ I DNO: 2. The antibody can detect the expression difference of the murine breast carcinoma cell 4T1 and DCF1 in the normal murine mammary tissue; the prepared polyclonal antibody can detect protein expression reduction of DCF1 after inhibiting cancer cell of 4T1 by JNKinhibitor II, therefore, which shows that the polyclonal antibody can be used for detecting the expression level of the DCF1 of the murine breast cancer at different states.

Description

technical field [0001] The invention relates to a novel mouse DCF1 specific polyclonal antibody and a preparation method thereof. technical background [0002] Dendritic cell factor 1 (DCF1), also known as transmembrane protein 59 (TMEM59), was first discovered in dendritic cells of the immune system and is a single transmembrane protein with a relatively common expression range. . According to UniProt's annotation information, DCF1 is mainly located on the Golgi apparatus. At present, there are relatively few reports on the function of this protein, and its function may be related to the transport of substances. Some reports and articles have shown that DCF1 and its homologous protein TMEM59-like can inhibit the transport of amyloid precursor protein APP to the cell membrane and further cleavage, and APP is related to the formation of Alzheimer's disease (AD), indicating that DCF1 has some connection with neurodegenerative diseases. [0003] According to the informat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24
Inventor 文铁桥程华
Owner SHANGHAI UNIV
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