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In-vitro expression method of pear S7-RNase protein and preparation method of polyclonal antibody of pear S7-RNase protein

A polyclonal antibody and in vitro expression technology, applied in the field of bioengineering, can solve problems such as high-titer targeted antibodies

Inactive Publication Date: 2018-06-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, S-RNase is an important factor affecting pear self-incompatibility, and there is no specific antibody with high potency and good specificity on the market. The role in sexual response and other related biological functions, this patent intends to prepare rabbit-derived anti-pear S 7 - RNase protein antibody

Method used

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  • In-vitro expression method of pear S7-RNase protein and preparation method of polyclonal antibody of pear S7-RNase protein
  • In-vitro expression method of pear S7-RNase protein and preparation method of polyclonal antibody of pear S7-RNase protein
  • In-vitro expression method of pear S7-RNase protein and preparation method of polyclonal antibody of pear S7-RNase protein

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Experimental program
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Effect test

Embodiment 1

[0063] Example 1: S 7 Construction and identification of -cutSignalP-pCold-TF recombinant plasmid

[0064]Take fresh Dangshansu pear flower style, plant total RNA extraction kit to extract style total RNA, reverse transcription with reverse transcription kit to obtain total cDNA; figure 1 As shown, analyze and remove pear S 7 -The signal peptide sequence of RNase gene, design primer, forward primer (S7-pCold-cutSignalP-Xho I-Lp) is 5'-CCG CTCGAG ATGTACGATTATTTTCAATTTACGCAGC-3' (the underline is the Xho I restriction site, the bold part is the start codon), the reverse primer (S7-pCold-cutSignalP-Xba I-Rp) is 5'-CTAG TCTAGA ATACTTAACATCGGCCGGGC-3' (the underline is the Xba I restriction site);

[0065] Use cDNA as a template for PCR amplification, and the PCR reaction system (total reaction system 50 μl) is: ddH 2 O 33μl, 1.5μl of upstream and downstream primers (10μm), 1μl of cDNA, 5μl of 10×KOD-Plus-PCR buffer, 5μl of 2mM dNTPs, 25mMMgSO 4 2μl, KOD-Plus-DNA polymerase...

Embodiment 2

[0067] Example 2: pear S 7 -Expression of RNase in Escherichia coli

[0068] 1. Obtaining the Expression of Pear S 7 -RNase recombinant expression strain

[0069] Bacteria successfully picked and sequenced were inoculated into 4 ml of LB liquid medium containing ampicillin (100 μg / ml), cultured with shaking at 37°C and 250 rpm overnight, and the S 7 -cutSignalP-pCold-TF recombinant expression vector extracted, take 1ng recombinant expression vector S 7 -cutSignalP-pCold-TF was transformed into Escherichia coli Rosetta (DE3), spread on a plate containing 100 μg / mL ampicillin to screen for recombinants, cultured at 37°C for overnight culture, and obtained recombinants expressing pear S 7 - RNase genetically engineered bacteria, randomly select a single colony for streak culture, inoculate a small amount of streak culture bacteria into 1ml LB (containing 100μg / ml ampicillin) liquid medium, shake culture at 37°C and 250rpm Overnight, then add 300 μl sterilized 20% (v / v) glycer...

Embodiment 3

[0072] Embodiment 3: recombinant pear S 7 -Isolation, purification and identification of RNase protein

[0073] Pear S 7 - RNase recombinant expression strains were inoculated into 100ml LB (containing 100 μg / ml ampicillin) liquid medium at a ratio of 1:50, and cultured overnight at 37°C with shaking at 250rpm to activate the recombinant expression strains. Transfer the activated recombinant expression strain to 300ml LB medium (containing ampicillin 100μg / mL) at a ratio of 1:50 for culture. The culture conditions are 37°C, 200rpm, shaking culture to OD 600 After 0.4-0.6 hours, put it on ice for 5 minutes quickly, then put it in a shaker at 15°C and let it stand for 40 minutes, finally add IPTG with a final concentration of 0.5mmol / L, and induce expression by shaking at 15°C and 240rpm for 24 hours . After the expression is completed, centrifuge at 4°C and 12000rpm for 10 minutes, discard the supernatant, collect the cell pellet, and use 20ml lysate (140mM sodium chloride, ...

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Abstract

The invention discloses an in-vitro expression method of a pear S7-RNase protein and a preparation method of a polyclonal antibody of the pear S7-RNase protein. The method comprises cloning a pear S7-RNase gene through primers, constructing an escherichia coli recombinant expression vector S7-cutSignalP-pCold-TF, transferring the recombinant expression vector S7-cutSignalP-pCold-TF into escherichia coli Rosetta (DE3) to construct a recombinant strain for expressing the pear S7-RNase protein, expressing the recombinant strain, purifying the recombinant pear S7-RNase protein through nickel column affinity chromatography purification and preparing a pear S7-RNase polyclonal antibody through the purified recombinant pear S7-RNase protein as an antigen through a conventional polyclonal antibodypreparation method. The method realizes in-vitro expression of the pear S7-RNase protein, prepares the high potency and good specificity pear S7-RNase polyclonal antibody and fully meets the needs ofrelated experiments.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a pear S 7 -In vitro expression method of RNase protein and preparation method of polyclonal antibody thereof. Background technique [0002] Pear self-incompatibility is one of the mechanisms that limit self-fertilization in angiosperms, which prevents the normal growth of pollen tubes with the same genotype in the pistil and completes fertilization. The self-incompatibility of pear is the self-incompatibility reaction of gametophyte based on S-RNase, and many fruit trees, such as apple, apricot, and plum, show this phenomenon. Since pear tree self-pollination cannot bear fruit, pollination trees must be configured in production, or artificial pollination can ensure fruit setting, which increases the production cost of farmers. Therefore, the research on the mechanism of pear self-incompatibility has both theoretical and practical value. In the past 30 years, a lot of r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/70C07K16/40
CPCC07K16/40C07K2317/20C12N9/22C12N15/70
Inventor 张绍铃汤超吴巨友王鹏施冬青
Owner NANJING AGRICULTURAL UNIVERSITY