CN107217066A

CN107217066A - The method and its application of DNA fragmentation and the recombinant vector containing the fragment, structure recombinant vector

Info

Publication number: CN107217066A
Application number: CN201710385597.XA
Authority: CN
Inventors: 赵臣; 赵阳; 赵佳琪; 袁忠海; 侯毅鞠; 李广庆
Original assignee: Jilin Medical College
Current assignee: Jilin Medical College
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2017-09-29

Abstract

The invention discloses DNA fragmentation, it is made up of the base sequence shown in the SEQ ID NO.1 in sequence table.The invention also discloses the recombinant vector containing exogenous polynucleotide, initial carrier and HSP70 MLAA34 fusions are included.The invention also discloses the method for building the recombinant vector containing exogenous polynucleotide, the genes of HSP 70 containing the base sequence shown in SEQ ID NO.4 are obtained using the primer sequence amplification shown in the SEQ ID NO.2 and SEQ ID NO.3 in sequence table, the genes of MLAA 34 containing the base sequence shown in SEQ ID NO.7 are obtained using the primer sequence amplification shown in the SEQ ID NO.5 and SEQ ID NO.6 in sequence table, using the genes of HSP 70 and the genes of MLAA 34 as template, utilize the primer sequence shown in the SEQ ID NO.6 in the SEQ ID NO.2 and sequence table in sequence table, amplification obtains the HSP70 MLAA34 fusions shown in the SEQ ID NO.1 in sequence table, by on HSP70 MLAA34 Fusion gene constructions to initial carrier to obtain recombinant vector.The invention also discloses the transfectional cell for including HSP70 MLAA34 fusions.

Description

DNA fragmentation and the recombinant vector containing the fragment, the method for building recombinant vector and its Using

Technical field

The present invention relates to biomedicine field, and in particular to DNA fragmentation is recombinated with the recombinant vector containing the fragment, structure The method and its application of carrier.

Background technology

Acute leukemia is a kind of relatively common Malignancy, is the lethal one of the main reasons of the mankind. In recent years, with the development of combined chemotherapy and HSCT technology so that leukaemia and other hematological system tumors Prognosis constantly improves, and can not thoroughly be removed yet with the microresidual disease (MRD) of tumour, recurrence rate and the death rate It is higher.

Research finds that nucleic acid vaccine can excite leukaemic's specific immune response, removes tumors remaining after chemotherapy Cell, is to be expected to overcome one of MRD method.Padua etc. is by the PML-RAR alpha fusion genes of mankind's progranulocyte leukemia Merged with the C fragment sequences of tetanus toxin gene, it was demonstrated that the nucleic acid vaccine using fusion protein as specific target spot can be notable Improve the survival rate of infected animal.MLAA-34 genes are closely related with acute monocytic leukemia (AML-M5) generation One new anti-apoptotic molecule, MLAA-34 down regulation of gene expression can significantly inhibit the increment of U937 cells in vitro, while can lure The apoptosis of leukaemia is sent out, and it is closely related with the poor prognosis such as AML-M5 low survival rate, it is the new swollen of acute leukemia The target gene of tumor markers and Gene immunotherapy.MLAA-34 albumen is intracellular protein, how effective submission MLAA-34 albumen To leukaemia's film surface so as to which successive induction body produces specific cellular immunity and as effector cell's cytotoxic T cell (CTL) targeted molecular, is that can vaccine play the key of leukemia resisting action.The generation of HSP70 and tumour, development, tumour Prognosis of immune and tumour etc. has substantial connection, and it has antigen submission function, can pass the antigenic information in tumour cell It is handed on cell membrane, and then induces body to produce specificity antineoplastic immunity response, and the immune response of its induction can not be by MHC Limitation, can play good antineoplastic immune effect, so as to play a part of killing tumour cell.

In addition, being received much concern always on the safety issue that can nucleic acid vaccine be incorporated into host genome.Research Show, immunization wayses have a significant impact to the integration security of DNA vaccination, immunized by electroporation mode can cause genome conformity to show As, and intramuscular injection and particle gun (gene gun) immunization wayses are safer, the DNA of nucleic acid vaccine is external to dye Additional sub form is present, rather than is incorporated on host chromosome DNA.

The content of the invention

One of goal of the invention of the present invention is to provide one group of DNA fragmentation.

The two of the goal of the invention of the present invention, which are to provide, includes the recombinant vector of above-mentioned DNA fragmentation.

The three of the goal of the invention of the present invention are to provide the method for building above-mentioned recombinant vector.

The four of the goal of the invention of the present invention are to provide the preparation that gene vaccine is carried out by above-mentioned recombinant vector.

The technical scheme that the present invention is provided is：

DNA fragmentation, is made up of the base sequence shown in the SEQ ID NO.1 in sequence table.

Recombinant vector containing exogenous polynucleotide, includes initial carrier and HSP70-MLAA34 fusions；

Wherein, the fusion includes the DNA pieces of the base sequence composition shown in the SEQ ID NO.1 in sequence table Section.

Preferably, the initial carrier is pIRES2-EGFP plasmids.

The method for building the recombinant vector containing exogenous polynucleotide, including：

Step 1: being expanded using the primer sequence shown in the SEQ ID NO.2 and SEQ ID NO.3 in sequence table by PCR Increasing obtains the HSP-70 genes containing the base sequence shown in SEQ ID NO.4；

Contained using the primer sequence shown in the SEQ ID NO.5 and SEQ ID NO.6 in sequence table by PCR amplifications There are the MLAA-34 genes of the base sequence shown in SEQ ID NO.7；

Step 2: using HSP-70 genes and MLAA-34 genes as template, utilizing the SEQ ID NO.2 and sequence in sequence table The primer sequence shown in SEQ ID NO.6 in list, the SEQ ID NO.1 institutes obtained in sequence table are expanded by SOE-PCR The HSP70-MLAA34 fusions shown, and it is contained EcoRI and BamHI restriction enzyme sites；

Step 3: will be carried on the HSP70-MLAA34 Fusion gene constructions described in step 2 to initial carrier with obtaining restructuring Body.

Preferably, the initial carrier is pIRES2-EGFP plasmids.

Preferably, including recombinant vector as claimed in claim 3 and U937 cells.

Preferably, its transfection method includes as follows：

Take U937 cells to be cultivated and collect the cell of exponential phase to be inoculated with, two parts of culture mediums are separately taken, at it Transfection reagent is added in middle a culture medium, the recombinant vector containing HSP70-MLAA34 fusions is added in another, Two parts of culture mediums are well mixed, the transfection composite after being well mixed is added in cell, is well mixed, cell is placed in 37 DEG C, 5%CO₂Incubator in cultivate 18~48 hours；Wherein, interchangeable fresh culture after transfecting 4~6 hours.

Preferably, the transfectional cell carries out transfection method including as follows：

Step a, U937 cell culture is taken in the culture mediums of RPMI 1640 containing 10% hyclone, 37 DEG C, 5%CO₂Training Support, change liquid 1 time within every 2~3 days, the cell in growth period of taking the logarithm is tested；

Step b, collection U937 cells simultaneously adjust cell concentration to 2 × 10⁵/ mL, is inoculated in 96 porocyte culture plates, often Hole adds the μ L of U937 cells 100；

Step c, dilute 0.8 μ g with 50 μ L Opti-MEM culture mediums and contain HSP70-MLAA34 fusions restructuring load Body, gently 3~5 mixings of pressure-vaccum；

Step d, gently reverse mixing transfection reagent, dilute 2.0 μ L's with 50 μ L Opti-MEM culture mediums LipofectamineTM3000 transfection reagents, gently 3~5 mixings of pressure-vaccum, stand 5 minutes at room temperature；

Step e, the mixing step c culture containing recombinant vector and the step d transfection reagent, gently pressure-vaccum 3~5 mixings, stand 20 minutes at room temperature, obtain transfection composite；

Step f, the transfection composite is added in 24 porocyte plates, 100 μ L/ holes, front and rear jog cell plates mixing Uniformly；

Step g, cell plates are placed in 37 DEG C, 5%CO₂Cultivated 18~48 hours in incubator；Wherein, transfect 4~6 hours Interchangeable fresh culture afterwards.

Gene vaccine is prepared using including described recombinant vector, the recombinant vector includes the SEQ ID in sequence table The DNA fragmentation of base sequence composition shown in NO.1.

The present invention is had the advantage that compared with prior art：

1st, can be by antigen presenting cell or body tissue cellular uptake after nucleic acid vaccine is imported in host, table in the cell Up to proteantigen, body is stimulated to produce cellular immunity and humoral immunity, immanoprotection action is good；

2nd, by SOE-PCR technologies when designing primer, overlap can be any site in gene order, be not required to Consider position of fusion and its neighbouring special sequence, bridge is used as by the primer of specific overlapping complementary sequences, so that PCR Amplified production end forms the overlapping chain of identical, and pcr amplified fragment is stitched together by the extension of overlapping chain, so that by two Individual target gene is merged, more simple and efficient compared to the digestion of restriction enzyme and the connection of ligase.

Brief description of the drawings

Fig. 1 is that SOE-PCR methods expand HSP70-MLAA34 fusions through 1.0% agarose gel electrophoresis qualification figure；Its In, 1 is molecular weight marker, and 2 be HSP70-MLAA34 fusions, and 3 be HSP70 genes, and 4 be MLAA34 genes.

Fig. 2 is pIRES2-HSP70-MLAA34 recombinant plasmids through 1.0% agarose gel electrophoresis qualification figure；Wherein, 1 is Molecular weight marker, 2 be pIRES2-HSP70-MLAA34 recombinant plasmids, and 3 be double digestion qualification result, and 4 be PCR qualification results.

Fig. 3 is checking gene gel electrophoresis images；Wherein, 1 is molecular weight marker, and 2 be pIRES2-EGFP plasmid transfections U937 cells, 3 be pIRES2-HSP70-MLAA34 Transfected Recombinant Plasmid U937 cells, and 4 be checking gene transfection U937 cells.

Fig. 4 is that purpose albumen detects figure in U937 cell inner expressions.

Fig. 5 is purpose albumen in U937 cell inner expression imaging analysis figures.

Embodiment

The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text Word can be implemented according to this.

" recombinant vector " in the present invention refers to the expression vector for being connected to gene, can be used interchangeably " recombinant plasmid ", " recombinant vector " and " nucleic acid vaccine ".

Experiment material and instrument

1st, plasmid and cell

U937 cells are purchased from Chinese Academy of Sciences's Shanghai cell bank；PIRES2-EGFP plasmids are purchased from addgene companies.

2nd, animal

BALB/c mouse [no-special pathogen (SPF) level, female, 6~8 week old] are purchased from the magnificent experimental animal skill of Beijing dimension tonneau Art Co., Ltd

3rd, reagent, enzyme and medicine

Tetrazolium bromide (MTT) reagent, restriction enzyme (I/BamH of EcoR I) are purchased from NEB companies of the U.S.；Total RNAs extraction is tried Agent box, endotoxin-free plasmid extraction kit, reverse transcription PCR (RT-PCR) kit, T4DNA ligases, glue reclaim purifying and The kits such as EUSA (ELISA) are purchased from Sangon Biotech (Shanghai) Co., Ltd.；Hyclone, RPMI 1640 (high sugar) culture medium is purchased from Hyclone companies of the U.S.；The conventional reagents such as absolute ethyl alcohol, chloroform are domestic analysis It is pure.

Embodiment

First, genetic recombination builds pIRES2-HSP70-MLAA34 recombinant plasmids：

1st, design of primers：

EcoR I is chosen according to Plasmid pIRES 2-EGFP multiple cloning sites situation and the restriction enzyme sites of BamH I carry out gene gram It is grand, MLAA-34 genes (GenBank：AY288977.2) amplified fragments theoretical length is 1014bp, HSP70 genes (GenBank： NM_005345.5) amplified fragments theoretical length is 1927bp, utilizes the primer and sequence in sequence table shown in SEQ ID NO.3 Design of primers overlap GGCGGCGGCGGCGGC in table shown in SEQ ID NO.5, primer is by giving birth to work bioengineering (Shanghai) Limited company synthesizes.

2nd, the structure of recombinant plasmid

Step 1: U937 cell culture is in the culture mediums of RPMI 1640 containing 10% hyclone, 37 DEG C, 5%CO₂, receive Collection U937 cells simultaneously adjust cell concentration to 2 × 10⁵/ mL, is inoculated in 96 porocyte culture plates, adds the μ of U937 cells 100 per hole The target cell that L is tested as splenic lymphocytes killing activity.

Step 2: HSP-70 gene magnifications and gene are extracted

Collect the U927 cells of culture, Trizol method extracted total RNAs, reverse transcription synthesis complementation RNA (cRNA), using following Primer enters the DNA fragmentation that performing PCR amplification obtains the base sequence in sequence table shown in SEQ ID NO.4, as HSP-70 genes：

Upstream end primer：

5 '-GCGAATTCATGAAAAAAATGCCTTTGTTTAGT-3 ' (the nucleosides in sequence table shown in SEQ ID NO.2 Acid sequence)；

Downstream primer：

5 '-GGCGGCGGCGGCGGCTCAAGGGGCCGTTTTCTTCAAG-3 ' are (in sequence table shown in SEQ ID NO.3 Nucleotide sequence)；

PCR reaction conditions：94 DEG C, 5min；94 DEG C, 30s；55 DEG C, 30s；72 DEG C, 60s, totally 30 circulations；Last 72 DEG C, 5min；Pcr amplification product is identified through 1.0% agarose gel electrophoresis, with glue reclaim kit recovery purifying PCR primer；

Step 3: MLAA-34 gene magnifications and gene are extracted

Collect the U927 cells of culture, Trizol method extracted total RNAs, reverse transcription synthesis complementation RNA (cRNA), using following Primer enters the DNA fragmentation that performing PCR amplification obtains the base sequence in sequence table shown in SEQ ID NO.7, as MLAA-34 bases Cause：

Upstream end primer：

5 '-GGCGGCGGCGGCGGCATGGCCAAAGCCGCGGCGATC-3 ' are (in sequence table shown in SEQ ID NO.5 Nucleotide sequence)；

Downstream primer：

5 '-CGGGATCCCTAATCTACCTCCTCAATG GTG-3 ' (the nucleotides in sequence table shown in SEQ ID NO.6 Sequence)；

PCR reaction conditions：94 DEG C, 5min；94 DEG C, 30s；55 DEG C, 30s；72 DEG C, 60s, totally 30 circulations；Last 72 DEG C, 5min；Pcr amplification product is identified through 1.0% agarose gel electrophoresis, with glue reclaim kit recovery purifying PCR primer.

Step 4: SOE-PCR methods expand HSP70-MLAA34 fusions：

By the PCR primer of recovery purifying, using MLAA-34 genes and HSP-70 genes as template, carried out using following primer SOE-PCR amplifications obtain the DNA fragmentation of the base sequence in sequence table shown in SEQ ID NO.1, and as HSP70-MLAA34 melts Close gene：

Upstream end primer：

Downstream primer：

SOE-PCR reaction conditions：94 DEG C, 5min；94 DEG C, 30s；58 DEG C, 30s；72 DEG C, 60s, totally 30 circulations；Finally 72 DEG C, 5min；SOE-PCR amplified productions are identified through 1.0% agarose gel electrophoresis, with glue reclaim kit recovery purifying SOE- PCR primer, experimental result is as shown in Figure 1；

Step 5: the structure of eukaryotic expression vector pIRES 2-HSP70-MLAA34 recombinant plasmids：

Extract Plasmid pIRES 2-EGFP, with the HSP70-MLAA34 fusions fragment of recovery carry out respectively EcoR I/ The double digestions of BamH I, 37 DEG C, 1.5h, digestion products are through agarose electrophoresis recovery purifying, using T4DNA ligases by HSP70- MLAA34 fusions are subcloned into pIRES2-EGFP plasmids, and 16 DEG C of connections are stayed overnight, and build pIRES2-HSP70-MLAA34 Recombinant plasmid；Connection product is converted into Escherichia coli (E.coli) DH5 α competent cells, and in kalamycin resistance LB cultures Coated plate culture on base；By the positive transformants clone grown on kalamycin resistance LB culture mediums numbering, with oese picking gram Longzi (should not choose completely) 37 DEG C of incubated overnights in 3mL LB fluid nutrient mediums, next day extracts pIRES2-HSP70- MLAA34 plasmids go forward side by side performing PCR amplification and digestion identification, picking positive colony bacterium, after amplification extract recombinant plasmid enter performing PCR and The double digestions of I/BamH of EcoR I are identified, are identified through 1.0% agarose electrophoresis, and experimental result is as shown in Figure 2.

2nd, cell-based assay

Step 1: cell culture

U937 cell culture is taken in the culture mediums of RPMI 1640 containing 10% hyclone, 37 DEG C, 5%CO₂Culture, every 2 Change within~3 days liquid 1 time, the cell in growth period of taking the logarithm is tested.

Step 2: recombinant vector transfects U937 cells

(1) U937 cells are collected and cell concentration are adjusted to 2 × 10⁵/ mL, is inoculated in 96 porocyte culture plates, per hole Plus the target cell that the μ L of U937 cells 100 are tested as splenic lymphocytes killing activity；

(2) 0.8 μ g recombinant vectors are diluted with 50 μ L Opti-MEM, gently 3~5 mixings of pressure-vaccum；

(3) gently overturn and mix transfection reagent, dilute 2.0 μ L's with 50 μ L Opti-MEM LipofectamineTM3000 transfection reagents, gently 3~5 mixings of pressure-vaccum, stand 5min at room temperature；

(4) (2) and (3) are mixed, gently 3~5 mixings of pressure-vaccum, stand 20min at room temperature；

(5) transfection composite is added in 24 porocyte plates, 100 μ L/ holes, and front and rear jog cell plates are well mixed；

(6) cell plates are placed in 37 DEG C, 5%CO₂Cultivate interchangeable fresh after 18~48hrs, 4~6hrs of transfection in incubator Culture medium.

Step 3: target gene mRNA is detected in U937 cell inner expressions level

U937 cells 1 × 10 two days later are taken before transfection and transfected respectively⁷, add 1mL Trizol reagents, 0.2mL chlorine Use 0.5mL isopropanol precipitating RNA after imitative extracting, Aspirate supernatant, then use DEPC after washing precipitation, natural airing with 75% ethanol Water dissolves and quantified；By total mRNA reverse transcriptions it is cDNA according to Reverse Transcriptase kit specification, ultraviolet specrophotometer determines it Concentration；Design primer is sense primer：5 '-CCCCATCATCAGCGGACTG-3 ' (shown in the SEQ ID NO.8 in sequence table), Anti-sense primer：5 '-CTTCTGTTGGGGGTTCTTT-3 ' (shown in the SEQ ID NO.9 in sequence table), enter by template of cDNA Performing PCR amplification is verified gene (shown in the SEQ ID NO.10 in sequence table) to determine target gene in the intracellular tables of U937 Reach, checking gene is the part in purpose gene, PCR reaction systems include 2.5 μ L 10 × buffer, 2.5 μ L dNTPs, 1.0 μ g cDNA templates, 0.1 μ L Taq enzymes (5U/ μ L), each 1 μ L of upstream and downstream primer, plus ddH₂O to 25 μ L；

PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C, 30s；62 DEG C, 30s,；72 DEG C, 30s；Circulation 30 times, then 72 DEG C of extension 10min, pcr amplification product is analyzed and identified with 1% agarose electrophoresis, Genius gel electrophoresis images analysis systems, Experimental result is as shown in Figure 3.

Step 4: destination protein is in U937 cell inner expressions level and protein active detection (western-blot)

Transfect after 48h, collect cell, add the μ L RIPA (1%PMSF) of lysate 150, blow and beat cell, be collected into centrifugation Guan Zhong, ice bath 30min, 12000r/min centrifugation 10min, extracts total protein, takes supernatant to add 2XSDS sample-loading buffers, boil Supernatant is taken to do on SDS-PAGE electrophoresis, electrotransfer to pvdf membrane after 5min, centrifugation, with MLAA-34 monoclonal antibodies as primary antibody, The goat anti-mouse igg of horseradish peroxidase as secondary antibody, after ECL colour developings in imaging of biomolecules instrument imaging analysis, As a result as shown in Figure 4, Figure 5.

3rd, integral level is detected

Step 1: BALB/c mouse is immunized in recombinant vector

DNA is largely prepared and purified and (carried out by kit specification) and adjusted with physiological saline to 1.0g/L； BALB/c mouse 40, are randomly divided into 4 groups, every group 10；Control group A group, experimental group B, C, D group are respectively in mouse both sides stock four-head Intramuscular intramuscular injection empty plasmid pIRES2, pIRES2-MLAA34, pIRES2-HSP70 and pIRES2-MLAA34-HSP70, every 100μg；24h before mouse is inoculated with every time, is pre-processed with the μ L of 0.25% Bupivacaine 100；0, carry out 3 same agent within 2,4 weeks Amount is immune.

Step 2: the preparation of splenic lymphocytes suspension

The 7th day after final immunization, mouse is put to death, 70% ethanol is put and soaks 3~5min, it is sterile to take spleen, it is placed in without tire In the culture mediums of RPMI 1640 of cow's serum, fat and fascia tissue are cut off, in being ground on 200 mesh stainless steel mesh screens, is resuspended in and contains In the Hank ' s liquid of 5% hyclone, 2000r/min centrifugation 5min, hypotonic lysis red blood cell, with containing 10% hyclone The culture mediums of RPMI 1640 are cleaned 3 times, and it is 1 × 10 to adjust concentration⁷/mL。

Step 3: detection and the result of splenic lymphocytes killing activity

Take the splenic lymphocytes suspension prepared in step 2, doubling dilution；Add spleen in each reacting hole of 96 porocyte culture plates The μ L of lymphocyte 100, make effect target ratio respectively 50：1、25：1、12.5：1 and 6.25：1 (respectively setting 3 multiple holes), in 37 DEG C, 5% CO₂Middle incubation 24h, determines every group of mouse specific lymphocyte killing activity；After reaction terminates, 20 μ L MTT works are added per hole Make liquid, 37 DEG C of reaction 4h discard MTT reaction solutions, DMSO (DMSO) 200 μ L, shaken at room temperature 10min, enzyme is added per hole Mark instrument and determine absorbance (A570) at 570nm.Splenic lymphocytes killing activity (%)=[1- (reacting hole A570- effector cells Control wells A570)/target cell control wells A570] × 100%.

After the immune mouse of nucleic acid vaccine packet, killing-efficiency of the sensitization splenic lymphocytes to U937 cells is detected, as a result It is shown in Table 1；Wherein, the killing-efficiency of nucleic acid vaccine D groups is apparently higher than A, B, C group, and difference is statistically significant (P ＜ 0.01)；And A, B, C group killing-efficiency compare, no significant difference (P ＞ 0.05).

The mouse spleen lymphocyte killing activity of table 1 (%)

*：P ＜ 0.01, are compared with D groups.

Step 4: detection and the result of cytokine levels

The splenic lymphocytes suspension of step 2 is added into 96 porocyte culture plates, per the μ L of hole 100 (setting 3 multiple holes), 37 DEG C, 5%CO₂72h is cultivated, with MLAA-34 albumen (20 μ g/mL) stimulator antigen of purifying；With concanavalin A (ConA, 10 μ g/ ML) it is positive control, unused antigenic stimulus hole is negative control, cultivates and supernatant is collected after 48h, ELISA method detection cell factor, Including interleukins (IL) -2, IL-4 and interferon-γ (IFN-γ) level (being operated in strict accordance with kit specification).

After the purified MLAA-34 albumen of splenic lymphocytes suspension is stimulated, cell in ELISA method detection Activity in Supernatant of Spleen Cell The factor IL-2, IL-4 and IFN-γ level, the results are shown in Table 2；Wherein, cell factor IL-2, IL-4 and IFN- of nucleic acid vaccine D groups γ levels are apparently higher than A, B, C group, and difference is statistically significant (P ＜ 0.01).B groups and the above-mentioned each cytokine levels of C groups are bright Aobvious to be higher than A groups, difference is statistically significant (P ＜ 0.01).Above-mentioned each cytokine levels compare between B groups and C groups, and difference is without system Meter learns meaning (P ＞ 0.05).

The mice spleen lymph suspension cytokine content of table 2 (pg/mL)

*：P ＜ 0.01, are compared with A groups；#：P ＜ 0.01, are compared with D groups.

5th, statistical procedures

Statistical analysis is carried out using the statistical softwares of SPSS 19.0, measurement data ± s compares to represent, between group uses variance Analysis, is that difference is statistically significant with P ＜ 0.05.

4th, interpretation of result

Nucleic acid vaccine make it is simple, using safety, be one of strategy of leukaemia immunization therapy.Nucleic acid vaccine imports host Can be by antigen presenting cell or body tissue cellular uptake after in vivo, expressing protein antigen, stimulates body to produce thin in the cell Born of the same parents are immunized and humoral immunity, and immanoprotection action is good.When building nucleic acid vaccine, author has used SOE-PCR technologies, by MLAA- 34 genes form MLAA34-HSP70 fusions together with HSP70 Gene Fusions.MLAA-34 genes and acute monokaryon are thin The generation of born of the same parents' leukaemia is closely related, and it, which expresses to lower, can significantly inhibit the propagation of U937 cells in vitro, while can induce white The apoptosis of blood disease cell, is the target gene of preferable acute leukemia immunization therapy.HSP70 participates in oncogene regulation and control and cell Propagation, with antigen submission function, energy intense stimulus immune system is directed to the immune response of tumour antigen, and then induces body production Raw specificity antineoplastic immunity response, the tumor vaccine based on HSP is the focus in current tumor vaccine research.

The key of SOE-PCR technologies essentially consists in the design of primer, and when designing primer, overlap can be gene sequence Any site on row, it is not necessary to consider position of fusion and its neighbouring special sequence.Pass through drawing for specific overlapping complementary sequences Thing, so that pcr amplification product end forms the overlapping chain of identical, is expanded PCR by the extension of overlapping chain as bridge Fragment assembly gets up, so that two target gene be merged, digestion and ligase compared to restriction enzyme Connection is more simple and efficient.Therefore, in fusion, the building process of mutant molecule, particularly struck in human somatic cell Remove, the research of vaccine, the generation of polyclonal antibody, and had a wide range of applications in plant genetic engineering.This research and design During primer using flexibility peptide gene GGCGGCGGCGGCGGC as overlap, between fusion protein MLAA-34 and HSP70 with Flexible peptide is connected, it is ensured that two protein fusion expressions and correct folding, MLAA-34 and HSP70 can all play its unique work( Can, so as to ensure that the immunocompetence of nucleic acid vaccine.Experiment shows that BALB/ is immunized in nucleic acid vaccine pIRES2-MLAA34-HSP70 After c mouse, sensitization splenic lymphocytes have stronger killing-efficiency to leukaemia, hence it is evident that higher than other experimental groups and control Group, illustrates that nucleic acid vaccine pIRES2-MLAA34-HSP70 can effectively stimulate body generation to be killed for the specificity of U937 cells Wound is acted on；In addition, cell factor IL-2, IL-4 and IFN-γ level substantially increase in sensitized mice spleen cell cultures liquid, explanation The nucleic acid vaccine can induce strong humoral immunity, enhance immune response of the body to tumour cell, with obvious immune Protective effect.

Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited In specific details and shown here as the legend with description.

SEQUENCE LISTING

<110>Jilin Medical College

<120>The method and its application of DNA fragmentation and the recombinant vector containing the fragment, structure recombinant vector

<130> 1

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 2994

<212> DNA

<213>Artificial sequence

<400> 1

gaattcatgg ccaaagcagc ggcgatcggc atcgacctgg gcaccaccta ctcctgcgtg 60

ggggtgttcc aacacggcaa ggtggagatc atcgccaacg accagggcaa ccgcaccacc 120

cccagctacg tggccttcac ggacaccgag cggctcatcg gggatgcggc caagaaccag 180

gtggcgctga acccgcagaa caccgtgttt gacgcgaagc ggctgatcgg ccgcaagttc 240

ggcgacccgg tggtgcagtc ggacatgaag cactggcctt tccaggtgat caacgacgga 300

gacaagccca aggtgcaggt gagctacaag ggggagacca aggcattcta ccccgaggag 360

atctcgtcca tggtgctgac caagatgaag gagatcgccg aggcgtacct gggctacccg 420

gtgaccaacg cggtgatcac cgtgccggcc tacttcaacg actcgcagcg ccaggccacc 480

aaggatgcgg gtgtgatcgc ggggctcaac gtgctgcgga tcatcaacga gcccacggcc 540

gccgccatcg cctacggcct ggacagaacg ggcaaggggg agcgcaacgt gctcatcttt 600

gacctgggcg ggggcacctt cgacgtgtcc atcctgacga tcgacgacgg catcttcgag 660

gtgaaggcca cggccgggga cacccacctg ggtggggagg actttgacaa caggctggtg 720

aaccacttcg tggaggagtt caagagaaaa cacaagaagg acatcagcca gaacaagcga 780

gccgtgaggc ggctgcgcac cgcctgcgag agggccaaga ggaccctgtc gtccagcacc 840

caggccagcc tggagatcga ctccctgttt gagggcatcg acttctacac gtccatcacc 900

agggcgaggt tcgaggagct gtgctccgac ctgttccgaa gcaccctgga gcccgtggag 960

aaggctctgc gcgacgccaa gctggacaag gcccagattc acgacctggt cctggtcggg 1020

ggctccaccc gcatccccaa ggtgcagaag ctgctgcagg acttcttcaa cgggcgcgac 1080

ctgaacaaga gcatcaaccc cgacgaggct gtggcctacg gggcggcggt gcaggcggcc 1140

atcctgatgg gggacaagtc cgagaacgtg caggacctgc tgctgctgga cgtggctccc 1200

ctgtcgctgg ggctggagac ggccggaggc gtgatgactg ccctgatcaa gcgcaactcc 1260

accatcccca ccaagcagac gcagatcttc accacctact ccgacaacca acccggggtg 1320

ctgatccagg tgtacgaggg cgagagggcc atgacgaaag acaacaatct gttggggcgc 1380

ttcgagctga gcggcatccc tccggccccc aggggcgtgc cccagatcga ggtgaccttc 1440

gacatcgatg ccaacggcat cctgaacgtc acggccacgg acaagagcac cggcaaggcc 1500

aacaagatca ccatcaccaa cgacaagggc cgcctgagca aggaggagat cgagcgcatg 1560

gtgcaggagg cggagaagta caaagcggag gacgaggtgc agcgcgagag ggtgtcagcc 1620

aagaacgccc tggagtccta cgccttcaac atgaagagcg ccgtggagga tgaggggctc 1680

aagggcaaga tcagcgaggc ggacaagaag aaggtgctgg acaagtgtca agaggtcatc 1740

tcgtggctgg acgccaacac cttggccgag aaggacgagt ttgagcacaa gaggaaggag 1800

ctggagcagg tgtgtaaccc catcatcagc ggactgtacc agggtgccgg tggtcccggg 1860

cctgggggct tcggggctca gggtcccaag ggagggtctg ggtcaggccc caccattgag 1920

gaggtagatg ggggcggagg ctccggcggc ggggggtctg ggggcggggg aagcatgaaa 1980

aaaatgcctt tgtttagtaa atcacacaaa aatccagcag aaattgtgaa aatcctgaaa 2040

gacaatttgg ccattttgga aaagcaagac aaaaagacag acaaggcttc agaagaagtg 2100

tctaaatcac tgcaagcaat gaaagaaatt ctgtgtggta caaacgagaa agaaccccca 2160

acagaagcag tggctcagct agcacaagaa ctctacagca gtggcctgct agtgacactg 2220

atagctgacc tgcagctgat agactttgag ggaaaaaaag atgtgaccca gatatttaac 2280

aacatcttga gaagacagat aggcactcgg agtcctactg tggagtatat tagtgctcat 2340

cctcatatcc tgtttatgct cctcaaagga tatgaagccc cacagattgc cttacgttgt 2400

gggattatgc tgagagaatg tattcgacat gaaccacttg ccaaaatcat cctcttttct 2460

aatcaattca gagatttctt taagtacgtg gagttgtcaa catttgatat tgcttcagat 2520

gcctttgcta ctttcaagga tttactaacc agacataaag tgttggtagc agacttctta 2580

gaacaaaatt acgacactat ttttgaagac tatgagaaat tgcttcagtc tgagaattat 2640

gttactaaga gacagtcttt aaagctgcta ggggagctga tcctggaccg tcacaacttt 2700

gccatcatga caaagtatat cagcaagccg gagaacctga aactcatgat gaacctcctt 2760

cgggataaaa gtcccaacat ccagtttgaa gcctttcatg tttttaaggt gtttgtggcc 2820

agtcctcaca aaacacagcc tattgtggag atcctgttaa aaaatcagcc caaactcatt 2880

gagtttctga gcagcttcca aaaagaaagg acggatgatg agcagttcgc tgacgagaag 2940

aactacttga ttaaacagat ccgagacttg aagaaaacgg ccccttgagg atcc 2994

<210> 2

<211> 32

<212> DNA

<213>Artificial sequence

<220>

<223>It is artificial synthesized, for use as the sense primer for the coded sequence for obtaining HSP-70 genes

<400> 2

gcgaattcat gaaaaaaatg cctttgttta gt 32

<210> 3

<211> 37

<212> DNA

<213>Artificial sequence

<220>

<223>It is artificial synthesized, for use as the anti-sense primer for the coded sequence for obtaining HSP-70 genes

<400> 3

ggcggcggcg gcggctcaag gggccgtttt cttcaag 37

<210> 4

<211> 1938

<212> DNA

<213>It is artificial synthesized

<220>

<223>The coded sequence of HSP-70 genes

<400> 4

gaattcatgg ccaaagcagc ggcgatcggc atcgacctgg gcaccaccta ctcctgcgtg 60

ggggtgttcc aacacggcaa ggtggagatc atcgccaacg accagggcaa ccgcaccacc 120

cccagctacg tggccttcac ggacaccgag cggctcatcg gggatgcggc caagaaccag 180

gtggcgctga acccgcagaa caccgtgttt gacgcgaagc ggctgatcgg ccgcaagttc 240

ggcgacccgg tggtgcagtc ggacatgaag cactggcctt tccaggtgat caacgacgga 300

gacaagccca aggtgcaggt gagctacaag ggggagacca aggcattcta ccccgaggag 360

atctcgtcca tggtgctgac caagatgaag gagatcgccg aggcgtacct gggctacccg 420

gtgaccaacg cggtgatcac cgtgccggcc tacttcaacg actcgcagcg ccaggccacc 480

aaggatgcgg gtgtgatcgc ggggctcaac gtgctgcgga tcatcaacga gcccacggcc 540

gccgccatcg cctacggcct ggacagaacg ggcaaggggg agcgcaacgt gctcatcttt 600

gacctgggcg ggggcacctt cgacgtgtcc atcctgacga tcgacgacgg catcttcgag 660

gtgaaggcca cggccgggga cacccacctg ggtggggagg actttgacaa caggctggtg 720

aaccacttcg tggaggagtt caagagaaaa cacaagaagg acatcagcca gaacaagcga 780

gccgtgaggc ggctgcgcac cgcctgcgag agggccaaga ggaccctgtc gtccagcacc 840

caggccagcc tggagatcga ctccctgttt gagggcatcg acttctacac gtccatcacc 900

agggcgaggt tcgaggagct gtgctccgac ctgttccgaa gcaccctgga gcccgtggag 960

aaggctctgc gcgacgccaa gctggacaag gcccagattc acgacctggt cctggtcggg 1020

ggctccaccc gcatccccaa ggtgcagaag ctgctgcagg acttcttcaa cgggcgcgac 1080

ctgaacaaga gcatcaaccc cgacgaggct gtggcctacg gggcggcggt gcaggcggcc 1140

atcctgatgg gggacaagtc cgagaacgtg caggacctgc tgctgctgga cgtggctccc 1200

ctgtcgctgg ggctggagac ggccggaggc gtgatgactg ccctgatcaa gcgcaactcc 1260

accatcccca ccaagcagac gcagatcttc accacctact ccgacaacca acccggggtg 1320

ctgatccagg tgtacgaggg cgagagggcc atgacgaaag acaacaatct gttggggcgc 1380

ttcgagctga gcggcatccc tccggccccc aggggcgtgc cccagatcga ggtgaccttc 1440

gacatcgatg ccaacggcat cctgaacgtc acggccacgg acaagagcac cggcaaggcc 1500

aacaagatca ccatcaccaa cgacaagggc cgcctgagca aggaggagat cgagcgcatg 1560

gtgcaggagg cggagaagta caaagcggag gacgaggtgc agcgcgagag ggtgtcagcc 1620

aagaacgccc tggagtccta cgccttcaac atgaagagcg ccgtggagga tgaggggctc 1680

aagggcaaga tcagcgaggc ggacaagaag aaggtgctgg acaagtgtca agaggtcatc 1740

tcgtggctgg acgccaacac cttggccgag aaggacgagt ttgagcacaa gaggaaggag 1800

ctggagcagg tgtgtaaccc catcatcagc ggactgtacc agggtgccgg tggtcccggg 1860

cctgggggct tcggggctca gggtcccaag ggagggtctg ggtcaggccc caccattgag 1920

gaggtagatt gaggatcc 1938

<210> 5

<211> 36

<212> DNA

<213>Artificial sequence

<220>

<223>It is artificial synthesized, for use as the sense primer for the coded sequence for obtaining MLAA-34 genes

<400> 5

ggcggcggcg gcggcatggc caaagccgcg gcgatc 36

<210> 6

<211> 30

<212> DNA

<213>Artificial sequence

<220>

<223>It is artificial synthesized, for use as the anti-sense primer for the coded sequence for obtaining MLAA-34 genes

<400> 6

cgggatccct aatctacctc ctcaatggtg 30

<210> 7

<211> 1026

<212> DNA

<213>It is artificial synthesized

<220>

<223>The coded sequence of MLAA-34 genes

<400> 7

gaattcatga aaaaaatgcc tttgtttagt aaatcacaca aaaatccagc agaaattgtg 60

aaaatcctga aagacaattt ggccattttg gaaaagcaag acaaaaagac agacaaggct 120

tcagaagaag tgtctaaatc actgcaagca atgaaagaaa ttctgtgtgg tacaaacgag 180

aaagaacccc caacagaagc agtggctcag ctagcacaag aactctacag cagtggcctg 240

ctagtgacac tgatagctga cctgcagctg atagactttg agggaaaaaa agatgtgacc 300

cagatattta acaacatctt gagaagacag ataggcactc ggagtcctac tgtggagtat 360

attagtgctc atcctcatat cctgtttatg ctcctcaaag gatatgaagc cccacagatt 420

gccttacgtt gtgggattat gctgagagaa tgtattcgac atgaaccact tgccaaaatc 480

atcctctttt ctaatcaatt cagagatttc tttaagtacg tggagttgtc aacatttgat 540

attgcttcag atgcctttgc tactttcaag gatttactaa ccagacataa agtgttggta 600

gcagacttct tagaacaaaa ttacgacact atttttgaag actatgagaa attgcttcag 660

tctgagaatt atgttactaa gagacagtct ttaaagctgc taggggagct gatcctggac 720

cgtcacaact ttgccatcat gacaaagtat atcagcaagc cggagaacct gaaactcatg 780

atgaacctcc ttcgggataa aagtcccaac atccagtttg aagcctttca tgtttttaag 840

gtgtttgtgg ccagtcctca caaaacacag cctattgtgg agatcctgtt aaaaaatcag 900

cccaaactca ttgagtttct gagcagcttc caaaaagaaa ggacggatga tgagcagttc 960

gctgacgaga agaactactt gattaaacag atccgagact tgaagaaaac ggccccttga 1020

ggatcc 1026

<210> 8

<211> 19

<212> DNA

<213>It is artificial synthesized

<220>

<223>It is artificial synthesized, for use as the anti-sense primer for the coded sequence for being verified gene

<400> 8

ccccatcatc agcggactg 19

<210> 9

<211> 19

<212> DNA

<213>It is artificial synthesized

<220>

<400> 9

cttctgttgg gggttcttt 19

<210> 10

<211> 350

<212> DNA

<213>It is artificial synthesized

<220>

<223>Verify the coded sequence of gene

<400> 10

ccccatcatc agcggactgt accagggtgc cggtggtccc gggcctgggg gcttcggggc 60

tcagggtccc aagggagggt ctgggtcagg ccccaccatt gaggaggtag atgggggcgg 120

aggctccggc ggcggggggt ctgggggcgg gggaagcatg aaaaaaatgc ctttgtttag 180

taaatcacac aaaaatccag cagaaattgt gaaaatcctg aaagacaatt tggccatttt 240

ggaaaagcaa gacaaaaaga cagacaaggc ttcagaagaa gtgtctaaat cactgcaagc 300

aatgaaagaa attctgtgtg gtacaaacga gaaagaaccc ccaacagaag 350

Claims

1.DNA fragments, it is characterised in that be made up of the base sequence shown in the SEQ ID NO.1 in sequence table.

2. the recombinant vector containing exogenous polynucleotide, it is characterised in that merged comprising initial carrier and HSP70-MLAA34 Gene；

Wherein, the fusion includes the DNA fragmentation of the base sequence composition shown in the SEQ ID NO.1 in sequence table.

3. recombinant vector as claimed in claim 2, it is characterised in that the initial carrier is pIRES2-EGFP plasmids.

4. build the method for the recombinant vector containing exogenous polynucleotide, it is characterised in that including：

Step 1: being expanded using the primer sequence shown in the SEQ ID NO.2 and SEQ ID NO.3 in sequence table by PCR To the HSP-70 genes containing the base sequence shown in SEQ ID NO.4；

Contained using the primer sequence shown in the SEQ ID NO.5 and SEQ ID NO.6 in sequence table by PCR amplifications The MLAA-34 genes of base sequence shown in SEQ ID NO.7；

Step 2: using HSP-70 genes and MLAA-34 genes as template, utilizing the SEQ ID NO.2 in sequence table and sequence table In SEQ ID NO.6 shown in primer sequence, expanded and obtained shown in SEQ ID NO.1 in sequence table by SOE-PCR HSP70-MLAA34 fusions, and it is contained EcoRI and BamHI restriction enzyme sites；

Step 3: by the HSP70-MLAA34 Fusion gene constructions described in step 2 to initial carrier to obtain recombinant vector.

5. the method for the recombinant vector containing exogenous polynucleotide is built as claimed in claim 4, it is characterised in that described original Carrier is pIRES2-EGFP plasmids.

6. include the transfectional cell of HSP70-MLAA34 fusions, it is characterised in that including restructuring as claimed in claim 3 Carrier and U937 cells.

7. transfectional cell as claimed in claim 6, its transfection method includes as follows：

Take U937 cells to be cultivated and collect the cell of exponential phase to be inoculated with, separately take two parts of culture mediums, wherein one Transfection reagent is added in part culture medium, the recombinant vector containing HSP70-MLAA34 fusions is added in another, by two Part culture medium is well mixed, and the transfection composite after being well mixed is added in cell, is well mixed, cell is placed in into 37 DEG C, 5%CO₂Incubator in cultivate 18~48 hours；Wherein, interchangeable fresh culture after transfecting 4~6 hours.

8. transfectional cell as claimed in claim 6, it is characterised in that the transfectional cell carries out transfection method including as follows：

Step a, U937 cell culture is taken in the culture mediums of RPMI 1640 containing 10% hyclone, 37 DEG C, 5%CO₂Culture, often Change within 2~3 days liquid 1 time, the cell in growth period of taking the logarithm is tested；

Step b, collection U937 cells simultaneously adjust cell concentration to 2 × 10⁵/ mL, is inoculated in 96 porocyte culture plates, adds per hole The μ L of U937 cells 100；

Step c, dilute 0.8 μ g with 50 μ L Opti-MEM culture mediums and contain HSP70-MLAA34 fusion recombinant vectors, gently 3~5 mixings are inhaled in featheriness；

Step e, the mixing step c culture containing recombinant vector and the step d transfection reagent, gently pressure-vaccum 3~5 It is secondary to mix, 20 minutes are stood at room temperature, transfection composite is obtained；

Step f, the transfection composite is added in 24 porocyte plates, 100 μ L/ holes, front and rear jog cell plates are well mixed；

Step g, cell plates are placed in 37 DEG C, 5%CO₂Cultivated 18~48 hours in incubator；Wherein, can after transfecting 4~6 hours Change fresh culture.

9. prepare gene vaccine using recombinant vector as claimed in claim 2 is included, it is characterised in that the recombinant vector bag DNA fragmentation containing the base sequence composition shown in the SEQ ID NO.1 in sequence table.