CN109912707B

CN109912707B - Lampetra lamprey immune protein LIP mutant capable of being used as tumor diagnosis marker

Info

Publication number: CN109912707B
Application number: CN201910170690.8A
Authority: CN
Inventors: 逄越; 李庆伟; 杨恺; 张珂嘉
Original assignee: Liaoning Normal University
Current assignee: Liaoning Normal University
Priority date: 2019-03-07
Filing date: 2019-03-07
Publication date: 2022-03-11
Anticipated expiration: 2039-03-07
Also published as: CN109912707A

Abstract

The invention discloses a lamprey immune protein LIP mutant capable of being used as a tumor marker, which is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown. The asparagine of the No. 286 amino acid in the lamprey immune protein LIP amino acid sequence is mutated into alanine, the unique property of specific positioning on tumor cells is still kept, but the killing function on the tumor cells is greatly reduced, the influence of the tumor morphology on the binding force with the tumor cells is avoided, the lamprey immune protein can be used as a tumor diagnosis marker, and the accuracy of tumor diagnosis is effectively improved.

Description

Lampetra lamprey immune protein LIP mutant capable of being used as tumor diagnosis marker

Technical Field

The invention relates to a tumor diagnosis marker, in particular to a lamprey immune protein LIP mutant which can be used as a tumor diagnosis marker.

Background

The total length of cDNA of the lamprey immune protein LIP (lamprey plum protein) is 1120bp, 313 amino acids are coded, the molecular weight of the protein is 34.2kD, and the protein belongs to secretory protein. The lamprey immune protein LIP has two functional structural domains, namely a Jacalin-like structural domain at the N end and an Aerolysin structural domain at the C end, and is positioned on the surface of a tumor cell by recognizing an N-sugar chain of a tumor cell GPI-APs and sphingomyelin in a tumor cell lipid raft micro-region; under the action of trace amount of lamprey immune protein LIP, tumor cells can bubble and leak contents in a short time, and finally cell death is caused, while Lamprey Immune Protein (LIP) has no killing effect on normal cells. Because Lamprey Immune Protein (LIP) has obviously different target cell specificities, relevant reports that lamprey immune protein LIP is used as a tumor diagnosis marker exist. But because the compound has strong killing activity on tumor cells, the compound destroys the tumor morphology, influences the binding force with the tumor cells and reduces the accuracy of tumor diagnosis.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a lamprey immune protein LIP mutant which can be used as a tumor diagnosis marker

The technical solution of the invention is as follows: a lamprey immune protein LIP mutant used as a tumor marker is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.

The invention mutates asparagine of No. 286 amino acid in the amino acid sequence of lamprey immune protein LIP into alanine, still retains the unique property of specific positioning on tumor cells, but greatly reduces the killing function on the tumor cells, avoids damaging the tumor morphology to influence the binding force with the tumor cells, can be used as a tumor diagnosis marker, and effectively improves the accuracy of tumor diagnosis.

Drawings

FIG. 1 is an electrophoresis diagram of lamprey immune protein LIP mutant according to an embodiment of the present invention.

FIG. 2 is a PI staining graph showing the killing of K562 cells by lamprey immune protein LIP mutant and wild lamprey immune protein LIP in real time according to the present invention.

FIG. 3 is a confocal image of laser scanning of lamprey immune protein LIP mutant localized on HeLa cell lipid rafts according to an embodiment of the present invention.

FIG. 4 is a photograph of an ultra-high resolution microscope showing the localization of lamprey immune protein LIP mutants to HeLa cell lipid rafts in accordance with an embodiment of the present invention.

Detailed Description

The lamprey immune protein LIP mutant capable of being used as a tumor marker is characterized in that the amino acid sequence is shown as SEQ ID NO: 1, asparagine of amino acid No. 286 in the lamprey immune protein LIP amino acid sequence is mutated into alanine.

Experiment:

preparation of lamprey immune protein LIP mutant

1. Is artificially synthesized as shown in SEQ ID NO: 1 to form lamprey immune protein LIP mutant, and transferring the mutant recombinant plasmid into an escherichia coli expression strain;

2. selecting monoclonal thallus, then placing the thallus in 5mL LB (adding ampicillin at a ratio of 1: 1000) culture medium for overnight culture (37 ℃, 120 rpm), transferring the bacterial liquid obtained by overnight culture (1: 100) into 500mL LB (containing 100μm mu M ampicillin) culture medium for continuous culture (37 ℃, 180 rpm), measuring the thallus concentration by an ultraviolet spectrophotometer, adding 0.5mM IPTG for induction when OD is approximately equal to 0.8, and continuously culturing the obtained bacterial liquid for 24h (16 ℃, 100 rpm);

3. centrifuging the bacterial solution (5000 rpm, 50min, 4 ℃), discarding the supernatant, retaining the obtained thalli, resuspending the thalli with a buffer (50 mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0), crushing the suspended bacterial solution with an ultrasonic bacteria breaker (ultra 5s, 10s, 40 min), centrifuging at high speed (12000 rpm, 30min, 4 ℃) to remove the fragments of the thalli, retaining the supernatant, passing the obtained supernatant through a Ni column: the supernatant was incubated with Ni gel (2 h, 4 ℃), the liquid was then removed, the Ni gel was washed with buffer 1, and the protein of interest was eluted with buffer 2 (50 mM Tris-HCl, 300mM NaCl, 70mM imidazole, pH 8.0);

4. further purifying the eluted protein with ion exchange column, and concentrating the protein to obtain lamprey immune protein LIP mutant (protein) with electrophoresis pattern as shown and storing at-80 deg.C.

Second, detection of killing condition of lamprey immune protein LIP mutant on MCF-7 and K562 cells

1. Cells MCF-7, K562 were cultured in 96-well plates.

2. Adding the lamprey immune protein LIP mutant and the wild lamprey immune protein LIP gradient into the mixture

0.5. mu.g and 1. mu.g, incubated at 37 ℃ for 20 min.

3. Dyes PI and Hoechst were added.

4. The results of the high content detection are shown in FIG. 2.

Compared with the wild lamprey immune protein LIP, the lamprey immune protein LIP mutant (LIPN 286A) provided by the invention has a significantly reduced tumor killing function.

Thirdly, detecting the labeling condition of the lamprey immune protein LIP mutant on MCF-7 and HeLa cells by a laser scanning confocal microscope and an ultrahigh resolution microscope

1. Cell plating: spreading HeLa cells in eight chambers, and culturing at 37 ℃ overnight;

2. and (3) incubation: absorbing culture medium, washing with PBS, adding lamprey immune protein LIP mutant, incubating at 37 deg.C for 2 hr with concentration of 1.2 μ g/well (prepared with PBS), setting a control group without protein, incubating under the same conditions,

3. fixing: sucking supernatant, washing with PBS, adding 4% paraformaldehyde 500 μ L, fixing for 20min, and washing with PBS twice;

4. and (3) sealing: blocking with 5% BSA for 5h at 37 ℃ (diluted in PBS);

5. a first antibody: adding primary antibody incubation (His mouse monoclonal antibody, control group is mouse source IgG) into the confining liquid at the ratio of 1:400, standing overnight at 4 ℃, and washing twice with PBS;

6. secondary antibody: adding donkey anti-mouse fluorescent secondary antibody according to the proportion of 1:600, incubating for 1h at 37 ℃ in the dark, and washing twice with PBS;

7. dyeing the core: adding DAPI dye according to the ratio of 1:2000, incubating for 20min in dark, and washing twice with PBS.

8. Performing laser scanning confocal microscope photographing and ultrahigh resolution microscope detection, respectively

Fig. 3 and 4. The result shows that the lamprey immune protein LIP mutant can be positioned on the surface of a tumor cell.

Sequence listing

<110> university of Liaoning teacher

<120> lamprey immune protein LIP mutant capable of being used as tumor diagnosis marker

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 313

<212> PRT

<213> Artificial sequence (Lampertra morii)

<220>

<222> (1)..(313)

<400> 1

Met Val Tyr Pro Thr Thr Leu His Ile Ile Gly Gly Gln Gly Gly Asn

1 5 10 15

Ala Phe Ser Phe Asn Gly Gln Glu Asn Ala Ala Thr Leu Gln Lys Leu

20 25 30

Ser Val Ser Val Gly Gly Trp Gln Val Arg Gly Val Gln Val Trp Leu

35 40 45

Thr Asp Gly Arg Arg Glu Thr Phe Gly Ala Met Asp Ser Ser Ala Lys

50 55 60

Glu Phe Glu Phe Glu Ser Gly Glu Phe Ile Lys Ser Leu Ser Leu Trp

65 70 75 80

Gly Asn Gly Ala Gly Thr Arg Leu Gly Ala Ile Lys Phe Ile Thr Ser

85 90 95

Arg Ser Arg Glu Phe Phe Ala Lys Met Thr Asp Trp Gly Leu Lys Thr

100 105 110

Glu Tyr Lys Ile Asp Val Gly Ser Gly Ile Cys Leu Gly Val Gln Gly

115 120 125

Arg Gly Gly Ser Asp Ile Asp Ser Met Gly Phe Ile Phe Ile Asn Ala

130 135 140

Ile Lys Ser Ser Val Ile Gln Asp Met Lys Tyr Pro Thr Met His Gln

145 150 155 160

Ile Leu Pro Asn Val Gln Met Glu Glu Ile Lys Glu Met Glu Tyr Lys

165 170 175

Asn Asp Thr Ser Ile Val Gln Ser Tyr Thr Phe Glu Ser Ser Lys Lys

180 185 190

Ile Ile Lys Lys Ser Ser Trp Ser Thr Thr Asn Lys Ile Glu Ser Thr

195 200 205

Phe Ser Leu Ser Val Lys Ala Gly Ile Pro Glu Val Met Glu Val Glu

210 215 220

Thr Gly Phe Ser Phe Thr Val Gly Ser Glu Ser Thr His Ala Val Glu

225 230 235 240

Glu Ser Glu Glu Lys Thr Glu Thr Leu Thr Phe Pro Val Thr Val Pro

245 250 255

Thr His Lys Thr Val Thr Val Val Ala Asn Ile Gly Arg Ala Asp Ile

260 265 270

Asp Leu Pro Tyr Thr Ala Leu Leu Arg Ile Thr Cys Val Ala Gly Ala

275 280 285

Ser Leu Asp Ala Pro Leu Ser Gly Ile Tyr Lys Gly Leu Thr Tyr Thr

290 295 300

Lys Met Thr Ala Val Ala Thr Glu Ser

305 310

Claims

1. A lamprey immune protein LIP mutant used as a tumor marker is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.