Recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and functional verification method of recombinant Escherichia coli

A technology for recombining Escherichia coli and scfv78, applied in the field of molecular biology, to achieve great application value, clear genome and genetic background, and easy transformation

Pending Publication Date: 2020-05-01

NANCHANG UNIV

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Problems solved by technology

[0008] The present invention aims at the technical defects of the prior art, and provides a recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 and its function verification m...

Abstract

The invention provides recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and a functional verification method of the recombinant Escherichia coli. The method comprises the following steps: firstly, connecting a recombinant single-chain antibody scFv78 encoding gene integrated with a Caspase-3 encoding gene to a pET302 vector through an enzyme digestion enzyme-linked approach, and converting obtained recombinant plasmid into Escherichia coli Nissle1917 so as to obtain a bacterial strain capable of expressing the Caspase-3 recombinant scFv78. The invention adopts the intracellular invasion and aggregation characteristics of the Escherichia coli Nissle1917 and the fixed-point targeting characteristic of the scFv78 on TEM1 positive cells; the human-derived toxin Caspase-3 protein site plays a role specifically, so that the TEM1 positive tumor cells are killed more accurately, and damage to normal cells of an organism is reduced. On the basis, the method utilizes recombinant bacteria to express the human-derived toxin Caspase-3 recombinant single-chain antibody scFv78 protein, and then the protein and MS1-TEM1 cells are co-incubated in an in-vitro environment, sothat the affinity and lethality of the to bacterial strain tumor cells are verified.

Application Domain

BacteriaMicroorganism based processes +3

Technology Topic

Ribosomal RNA GenesMolecular biology +12

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Examples

Experimental program(2)

Example Embodiment

[0034] Example 1

[0035] 1. Using pET302-78-Caspase-3 as a template and SEQ ID No. 3 and SEQ ID No. 4 as primers, PCR amplification obtains the 78-Caspase-3 fragment shown in SEQ ID No. 1.

[0036] Among them, 78 is a single-chain antibody that specifically recognizes TEM1 that has been screened in the laboratory and named 78; Caspase3 is an apoptosis-inducing factor. The gene sequence of Caspase3 fragment is obtained through GeneBank, and then it is handed over to the company (Nanjing GenScript) ) Carry out chemical synthesis of the whole sequence to obtain pUC57-78-Csapase3 recombinant plasmid or pET302-78-Caspase-3 recombinant plasmid. Furthermore, using pET302-78-Caspase-3 or pUC57-78-Csapase3 as a template and SEQ ID No. 3 and SEQ ID No. 4 as primers, the 78-Caspase shown in SEQ ID No. 1 was obtained by PCR amplification. 3 fragments.

[0037] 2. The obtained 78-Caspase-3 fragment and plasmid pET302 were digested with Xho I and AvrⅡ. After the digested product was purified, it was ligated into pET302 (SEQ ID No. 2) plasmid with T4 ligase, and the recombinant plasmid was named It is pET302-78-Caspase-3. The result is figure 1 Shown.

[0038] 3. Streak and inoculate E. coli Nissle1917 stored at -80°C on a non-resistant LB plate, culture overnight at 37°C; pick a single colony into 5mL LB, culture with shaking at 37°C for 12h; inoculate 100 at a ratio of 1:100 Culture in mL LB with shaking until the bacteria OD = 0.4; after ice bath for 20 minutes, centrifuge at 4℃ 3000rpm for 10min; wash the bacterial pellet twice with 1/10 volume of pre-cooled sterile deionized water, centrifuge at 4℃ 3000rpm for 10min; The cell pellet was washed again with 1/100 volume of pre-cooled 10% glycerol, and centrifuged at 3000 rpm at 4°C for 10 min; the cell pellet was resuspended in 1/100 volume of pre-cooled 10% glycerol to prepare E. coli Nissle 1917 competent Store at -80°C for later use.

[0039] 4. Transform the obtained recombinant plasmid pET302-78-Caspase-3 into E. coli Nissle1917, take E. coli Nissle1917 competent cells (100μL) and melt on ice, add the recombinant plasmid (2μL), tap the tube wall to mix, and place Incubate on ice for 30min; immerse the bacterial solution in a 42℃ water bath for 60-90s (without vibration), immediately place it on ice and let stand for 2min; add 900μL of SOC culture solution and incubate for 1h with shaking on a constant temperature shaker at 37℃( 220rpm); Centrifuge the bacterial solution at 4000rpm for 3min, discard the excess liquid, resuspend the bacterial cells with the remaining 100μL of medium, add all the bacterial solution to the ampicillin-resistant LB solid culture plate, and place it at 37°C and invert it overnight. The positive clones were screened, and recombinant E. coli Nissle1917 was obtained, named E.coli Nissle1917-pET302-78-Caspase-3.

[0040] 5. The 96-well plate was coated with 2% gelatin (37°C, 30min), and after the gelatin was shaken off, MS1-TEM1 cells and MS1 cells were connected to the plate (37°C, overnight growth). The scFv78 diluted with PBS was incubated with the cells (4°C, 2h), and then the secondary antibody was incubated at 4°C for 1h. The OD value was measured by color development, and the affinity of 78-Caspase-3 to TEM1 positive cells was detected by live cell ELISA. The experimental results are as figure 2 , image 3 As shown, the recombinant Escherichia coli E.coli Nissle1917-pET302-78-Caspase-3 of the present invention has good affinity and lethality to TEM1 positive cells, thereby proving that the recombinant bacteria of the present invention has an exact anti-tumor effect.

Example Embodiment

[0041] Example 2

[0042] The recombinant E. coli Nissle 1917 expressing Caspase-3 recombinant scFv78 was constructed by the following method:

[0043] 1) Connect the human toxin Caspase-3 recombinant single-chain antibody scFv78 coding gene fragment 78-Caspase-3 into the pET302 plasmid by enzyme digestion method to obtain the pET302-78-Caspase-3 recombinant plasmid;

[0044] 2) Transform the pET302-78-Caspase-3 recombinant plasmid into E. coli Nissle 1917 to obtain recombinant E. coli Nissle1917-pET302-78-Caspase-3, which is the recombinant scFv78 expressing Caspase-3 Recombinant E. coli Nissle 1917.

[0045] Wherein, in step 1), the human toxin Caspase-3 recombinant single-chain antibody scFv78 encoding gene fragment 78-Caspase-3 is a DNA fragment with a nucleotide sequence as shown in SEQ ID No.1.

[0046] Step 1) The human toxin Caspase-3 recombinant single-chain antibody scFv78 encoding gene fragment 78-Caspase-3 is based on the DNA fragments with nucleotide sequences such as SEQ ID No. 3 and SEQ ID No. 4 as primers, Amplified by PCR.

[0047] The pET302-78-Caspase-3 recombinant plasmid used in step 2) is obtained by first transferring the pET302-78-Caspase-3 recombinant plasmid obtained in step 1) into E. coli Top10, and then culture and amplification.

[0048] Step 1) The enzyme digestion enzyme linkage is double digestion with Xho I and AvrII. After the digestion product is purified, it is ligated into the pET302 plasmid using T4 ligase.

[0049] Step 2) includes: taking 100 μL of E. coli Nissle1917 competent cells to melt on ice, adding 2 μL of the pET302-78-Caspase-3 recombinant plasmid, mixing, and incubating on ice for 30 min. Immerse in a 42℃ water bath and heat shock for 60~90s, then immediately place it on ice and let it stand for 2min, then add 900μL of SOC broth to it, and incubate on a 37℃ constant temperature shaker with shaking at 22rpm for 1h, and then at 4000rpm Centrifuge the bacterial solution for 3 minutes, discard the excess liquid, resuspend the bacteria with the remaining 100μL of culture medium, add all the bacterial solution to the ampicillin-resistant LB solid culture plate, invert the culture overnight at 37℃, select positive clones , The recombinant E. coli Nissle1917-pET30 2-78-Caspase-3 is obtained, which is the recombinant E. coli Nissle 1917 expressing the Caspase-3 recombinant scFv78.

[0050] The E. coli Nissle1917 competent cells were prepared by the following method: E. coli Nissle1917 was streaked and inoculated on a non-resistant LB plate and cultured overnight at 37°C; a single colony was picked and inoculated in 5 mL of LB medium and shaken at 37°C Cultivate for 12h; inoculate 100mL LB medium according to the ratio of 1:100, shake culture until the bacteria OD value is 0.4; ice bath for 20min, then centrifuge at 4℃, 3000rpm for 10min; the bacterial pellet is pre-cooled with 1/10 volume Wash twice with sterile deionized water, centrifuge at 4°C, 3000rpm for 10min; wash the bacterial pellet again with 1/100 volume of precooled 10% glycerol, centrifuge at 4°C, 3000rpm for 10min; resuspend the bacterial pellet at 1 /100 volume of pre-cooled 10% glycerol to obtain the E. coli Nissle1917 competent cells.

[0051] The pET302 plasmid is a DNA molecule whose nucleotide sequence is shown in SEQ ID No.2.

[0052] Next, the following method A or B can be used to verify the tumor cell killing function of recombinant E. coli Nissle 1917:

[0053] Method A: Use the recombinant E.coli Nissle1917-pET302-78-Caspase-3 to express the human toxin Caspase-3 recombinant single chain antibody scFv78, and then combine the human toxin Caspase-3 recombinant single chain antibody scFv78 with The TEM1 positive cells were incubated together, and then the affinity of the human toxin Caspase-3 recombinant single chain antibody scFv78 and the TEM1 positive cells was detected by live cell ELISA.

[0054] Method B: Coat a 96-well plate with 2% gelatin at 37°C for 30 minutes, shake off the gelatin, connect MS1-TEM1 cells and MS1 cells to the plate, and grow overnight at 37°C; at 4°C, dilute with PBS After the human toxin Caspase-3 recombinant single-chain antibody scFv78 was incubated with the cells for 2 hours, and then the secondary antibody was incubated for 1 hour at 4°C, the OD value was measured by color development, and the human toxin Caspase-3 recombinant single-chain antibody was detected by live cell ELISA. The affinity of antibody scFv78 to the MS1-TEM1.

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reduce damage
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Recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and functional verification method of recombinant Escherichia coli

A technology for recombining Escherichia coli and scfv78, applied in the field of molecular biology, to achieve great application value, clear genome and genetic background, and easy transformation

AI-Extracted Technical Summary

Problems solved by technology

Abstract

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Examples

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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