CN109912707A - It can be used as the lamprey immune protein LIP mutant of diagnosing tumor marker - Google Patents
It can be used as the lamprey immune protein LIP mutant of diagnosing tumor marker Download PDFInfo
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- CN109912707A CN109912707A CN201910170690.8A CN201910170690A CN109912707A CN 109912707 A CN109912707 A CN 109912707A CN 201910170690 A CN201910170690 A CN 201910170690A CN 109912707 A CN109912707 A CN 109912707A
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Abstract
The present invention discloses a kind of lamprey immune protein LIP mutant that can be used as tumor marker, it is characterised in that amino acid sequence is as shown in SEQ ID NO:1.The asparagine mutation for being No. 286 amino acid in lamprey immune protein LIP amino acid sequence is alanine, still remain the peculiar property being specifically located on tumour cell, but greatly reduce the killing ability for tumour cell, it has avoided damage to shape of tumor and has influenced the binding force with tumour cell, it can be used as diagnosing tumor marker, effectively improve the accuracy of diagnosing tumor.
Description
Technical field
The present invention relates to a kind of diagnosing tumor marker, especially a kind of lamprey that can be used as diagnosing tumor marker is exempted from
Epidemic disease albumen LIP mutant.
Background technique
Lamprey immune protein LIP(lamprey Lee albumen), cDNA overall length is 1120bp, encodes 313 amino acid, egg
White matter molecular weight is 34.2kD, belongs to secreted protein.There are two functional domains for lamprey immune protein LIP tool, are N respectively
The Jacalin-like structural domain at end and the Aerolysin structural domain of C-terminal, pass through the N- sugar chain of tumor cell GPI-APs
And the sphingomyelins of tumour cell Lipid Rafts microcell makes lamprey immune protein LIP be located in tumor cell surface；Micro seven
Under gill eel immune protein LIP effect, short time inner tumour cell will occur to be bubbled, content phenomena such as leaking, and eventually lead to
Cell death, and lamprey immune protein (LIP) to normal cell without lethal effect.Because lamprey immune protein (LIP) has
There is visibly different target cell specificity, therefore has using lamprey immune protein LIP as the correlation report of diagnosing tumor marker
Road.But because it has strong killing activity for tumour cell, therefore shape of tumor is destroyed, affected and tumour cell
Binding force reduces the accuracy of diagnosing tumor.
Summary of the invention
The present invention is to solve above-mentioned technical problem present in the prior art, and providing one kind can be used as diagnosing tumor mark
Remember the lamprey immune protein LIP mutant of object
Technology technical solution of the invention is: a kind of lamprey immune protein LIP mutant can be used as tumor marker,
It is characterized in that amino acid sequence is as shown in SEQ ID NO:1.
The present invention is that the asparagine mutation of No. 286 amino acid in lamprey immune protein LIP amino acid sequence is
Alanine still remains the peculiar property being specifically located on tumour cell, but greatly reduces for tumour cell
Killing ability, avoided damage to shape of tumor and influenced the binding force with tumour cell, can be used as diagnosing tumor marker,
Effectively improve the accuracy of diagnosing tumor.
Detailed description of the invention
Fig. 1 is the electrophoretogram of lamprey immune protein LIP mutant of the embodiment of the present invention.
Fig. 2 is example lamprey immune protein LIP mutant in real time of the invention and LIP pairs of immune protein of wild type lamprey
The PI colored graph of K562 cell killing situation.
Fig. 3 is that the laser that lamprey immune protein LIP mutant of the embodiment of the present invention is positioned on HeLa cell Lipid Rafts is swept
Surface confocal picture.
Fig. 4 is that lamprey of embodiment of the present invention immune protein LIP mutant is positioned at the superelevation on HeLa cell Lipid Rafts point
Distinguish microscope photograph.
Specific embodiment
The lamprey immune protein LIP mutant that can be used as tumor marker of the invention, it is characterised in that amino acid sequence
Column are the asparagines of No. 286 amino acid in lamprey immune protein LIP amino acid sequence as shown in SEQ ID NO:1
Sport alanine.
Experiment:
One, prepares lamprey immune protein LIP mutant
1. the artificial synthesized amino acid sequence as shown in SEQ ID NO:1 forms lamprey immune protein LIP mutant, and takes prominent
Variant recombinant plasmid, is transferred to E. coli expression strains；
2. picking monoclonal thallus, it is subsequently placed at 5mL LB(1:1000 ratio and ammonia benzyl antibiotic is added) it carried out in culture medium
Night cultivates (37 DEG C, 120rpm), will be incubated overnight gained bacterium solution switching (1:100) and enters 500mL LB(containing 100m μM of ammonia benzyl antibiosis
Element) continue in culture medium to cultivate (37 DEG C, 180rpm), cell concentration is measured by ultraviolet specrophotometer, to
When OD ≈ 0.8,0.5mM IPTG is added and is induced, gained bacterium solution is continued into culture for 24 hours (16 DEG C, 100rpm)；
3. bacterium solution is centrifuged (5000rpm, 50min, 4 DEG C), supernatant is abandoned, the thallus retained utilizes buffer
(50mM Tris-HCl, 300mM NaCl, 10mM imidazoles, pH8.0) thallus is resuspended, using carrying out ultrasonic bacteria breaking instrument to suspension
Bacterium solution is crushed (super 5s stops 10s, 40min), high speed centrifugation (12000rpm, 30min, 4 DEG C), to remove bacterial chip,
Retain supernatant, resulting supernatant is crossed into Ni column: supernatant and Ni glue being first incubated for (2h, 4 DEG C), then removes wherein
Liquid is rinsed Ni glue with buffer 1, then uses buffer 2(50mM Tris-HCl, 300mM NaCl, 70mM imidazoles,
PH8.0) destination protein is eluted；
4. the above-mentioned albumen afforded is further purified using ion exchange column, gained albumen is concentrated, i.e.,
Obtain lamprey immune protein LIP mutant (albumen) of the present invention, electrophoretogram as shown, be subsequently stored in -80 DEG C it is spare.
Two, lamprey immune protein LIP mutant are to cell MCF-7 and K562 killing situation detection
1. cell MCF-7, K562 are cultivated in 96 orifice plates.
2. lamprey immune protein LIP mutant, which is added, with wild type lamprey immune protein LIP gradient is
0.5 μ g and 1 μ g, 37 DEG C of incubation 20min.
3. dyestuff PI and Hoechst is added.
4. high intension detection, as a result as shown in Figure 2.
Lamprey immune protein LIP mutant (LIPN286A) of the present invention compared to wild type lamprey immune protein LIP,
Tumor-killing function is substantially reduced.
Three, laser scanning co-focusing microscopes and ultrahigh resolution microscope detect lamprey immune protein LIP of the present invention
Label situation of the mutant to MCF-7 and HeLa cell
1. plating cells: HeLa cell being laid in eight company cells, 37 DEG C are incubated overnight；
2. being incubated for: culture medium is drawn, is rinsed with PBS, experimental group addition lamprey immune protein LIP mutant of the present invention, 37 DEG C
It is incubated for 2h, concentration is 1.2 holes μ g/ (being prepared with PBS), and a control group is separately arranged, does not add albumen, and incubation conditions are identical,
3. fixed: drawing supernatant, rinsed with PBS, 4% paraformaldehyde, 500 μ L is added, fixed 20min is washed twice with PBS；
4. closing: being diluted with 5% BSA, 37 DEG C of closing 5h(with PBS)；
5. primary antibody: primary antibody is added in confining liquid and is incubated for (His mouse monoclonal antibody, control group be mouse source IgG), ratio 1:400,4
DEG C overnight, PBS is washed twice；
6. secondary antibody: in 1:600 ratio, the anti-mouse fluorescence secondary antibody of donkey is added, 37 DEG C are protected from light incubation 1h, and PBS is washed twice；
7. contaminating core: DAPI dyestuff is added in 1:2000 ratio, is protected from light and is incubated for 20min, PBS is washed twice.
8. progress laser scanning co-focusing microscope is taken pictures and the detection of ultrahigh resolution microscope, as a result respectively such as
Shown in Fig. 3, Fig. 4.The result shows that lamprey immune protein LIP mutant of the invention can be positioned on tumor cell surface.
Sequence table
<110>Liaoning Normal University
<120>it can be used as the lamprey immune protein LIP mutant of diagnosing tumor marker
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 313
<212> PRT
<213>artificial sequence (Lampetra 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)
1. a kind of lamprey immune protein LIP mutant that can be used as tumor marker, it is characterised in that amino acid sequence is such as
Shown in SEQ ID NO:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910170690.8A CN109912707B (en) | 2019-03-07 | 2019-03-07 | Lampetra lamprey immune protein LIP mutant capable of being used as tumor diagnosis marker |
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| CN201910170690.8A CN109912707B (en) | 2019-03-07 | 2019-03-07 | Lampetra lamprey immune protein LIP mutant capable of being used as tumor diagnosis marker |
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| Publication Number | Publication Date |
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| CN109912707A true CN109912707A (en) | 2019-06-21 |
| CN109912707B CN109912707B (en) | 2022-03-11 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114711433A (en) * | 2022-03-09 | 2022-07-08 | 辽宁师范大学 | Application of lamprey LIP protein in preparation of food and medicine for treating obesity and improving cold resistance |
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| AU2001254623A1 (en) * | 2000-04-28 | 2001-11-12 | Novozymes A/S | Production and use of protein variants having modified immunogenicity |
| CN103554242A (en) * | 2013-10-23 | 2014-02-05 | 辽宁师范大学 | Liproteins, preparation method and application of liproteins in preparing medicament for preventing and treating tumor diseases |
| CN106589100A (en) * | 2016-12-14 | 2017-04-26 | 辽宁师范大学 | Angiogenesis-resistance lampetra japonica recombinant protein PR-1 and preparation method thereof |
| CN108169476A (en) * | 2017-12-22 | 2018-06-15 | 辽宁师范大学 | Using lamprey Lee albumen as the lesion detection kit of marker |
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2019
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2001254623A1 (en) * | 2000-04-28 | 2001-11-12 | Novozymes A/S | Production and use of protein variants having modified immunogenicity |
| CN103554242A (en) * | 2013-10-23 | 2014-02-05 | 辽宁师范大学 | Liproteins, preparation method and application of liproteins in preparing medicament for preventing and treating tumor diseases |
| CN106589100A (en) * | 2016-12-14 | 2017-04-26 | 辽宁师范大学 | Angiogenesis-resistance lampetra japonica recombinant protein PR-1 and preparation method thereof |
| CN108169476A (en) * | 2017-12-22 | 2018-06-15 | 辽宁师范大学 | Using lamprey Lee albumen as the lesion detection kit of marker |
Non-Patent Citations (7)
| Title |
|---|
| PANG, YUE等: "A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells", 《CELL COMMUNICATION AND SIGNALING》 * |
| XIAOYUAN CHI等: "Lamprey immune protein-1 (LIP-1) from Lampetra japonica induces cell cycle arrest and cell death in HeLa cells", 《FISH SHELLFISH IMMUNOL》 * |
| YUE PANG等: "Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells", 《CELL COMMUNICATION AND SIGNALING》 * |
| 于涛: "东北七鳃鳗Li protein的分子克隆、原核表达、抗体制备及组织分布研究", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
| 张珂嘉: "七鳃鳗LIP蛋白晶体结构解析和相关位点突变的活性鉴定", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 裴广盈: "重组七鳃鳗LIP分子对肿瘤细胞杀伤机制-基于细胞膜靶点的探究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》 * |
| 韩英伦: ""首届七鳃鳗免疫系统研究国际会议"在辽宁师范大学胜利召开"", 《遗传》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114711433A (en) * | 2022-03-09 | 2022-07-08 | 辽宁师范大学 | Application of lamprey LIP protein in preparation of food and medicine for treating obesity and improving cold resistance |
| JP7372713B2 (en) | 2022-03-09 | 2023-11-01 | 遼寧師範大学 | Use of lamprey LIP protein in the production of foods and drugs to treat obesity and improve cold tolerance |
| CN114711433B (en) * | 2022-03-09 | 2024-02-02 | 辽宁师范大学 | Application of lamprey LIP protein in preparation of medicines for treating obesity and improving cold resistance |
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| CN109912707B (en) | 2022-03-11 |
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