CN106562999B - The reagent set and recombinant influenza for treating tumour - Google Patents
The reagent set and recombinant influenza for treating tumour Download PDFInfo
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Abstract
The invention discloses the reagent sets and recombinant influenza for the treatment of tumour.The reagent set for the treatment of tumour provided by the present invention, is made of adriamycin and recombinant influenza；The recombinant influenza expresses following B1) or protein B2): B1) amino acid sequence be sequence 1 protein；B2) in the amino acid sequence shown in sequence 1 through substitution and/or be deleted and/or added one or several amino acid residues obtain it is with the same function as B1) derived from protein.It is demonstrated experimentally that reagent set and recombinant influenza of the invention can inhibit the growth of liver cancer and can extend the life span of animal, can be used to treat liver cancer.
Description
Technical field
The present invention relates to reagent sets and recombinant influenza that tumour is treated in biomedicine field.
Background technique
Primary carcinoma of liver is one of most common malignant tumour in China.The morbidity number height of the liver cancer in China ranks first in the world,
The morbidity population of nearly half is located at China in the world.Clinical treatment liver cancer often applies the means such as operation, radiotherapy, chemotherapy at present, but
It is difficult to eradicate liver cancer.Traditional Radiotherapy chemotherapy treatment method also makes the normal cell of human body while killing tumor cell
At huge injury.Therefore treatment liver cancer a great problem be, how efficiently, specific killing tumour cell while, reduce pair
The damage of normal cell tissue.
People H1N1 Influenza virus strain A/PR/8/34 (abbreviation PR8) is that one plant of chicken embryo adapts to Strain, can be had in chicken embryo
The duplication of effect ground.PR8 is sub-thread minus strand, segmented RNA, shares 8 independent RNA segment compositions, encodes 10 kinds of protein.Piece
The RNA polymerase that section 1-3 coding RNA relies on, 1 encoded polymerase subunit PB2 of segment, 2 encoded polymerase subunit PB1 of segment, piece
3 encoded polymerase subunit PA of section；Segment 4 encodes hemagglutinin HA, is a kind of surface glycoprotein with viral attachment infected with pass；Piece
5 encoding nuclear proteins NP of section, are the dominant structural moieties of viral RNA；6 encoding nerve propylhomoserin enzyme NA of segment, is a kind of coating sugar egg
It is white；Segment 7 encodes two kinds of matrix prote m1s and M2, is nonglycosylated structural proteins；Segment 8 encodes two kinds of non-structural proteins
NS1 and NS2.
Summary of the invention
The technical problem to be solved by the present invention is to how treat liver cancer.
In order to solve the above technical problems, present invention firstly provides the reagent sets for the treatment of tumour.
The reagent set for the treatment of tumour provided by the present invention, is made of adriamycin and recombinant influenza；The recombination
Influenza virus expresses HCKP, protein of the HCKP for following B1) or B2):
R1) amino acid sequence is the protein of sequence 1；
B2) by replacing and/or being deleted and/or added one or several amino in the amino acid sequence shown in sequence 1
Sour residue obtains with the same function as B1) derived from protein.
In above-mentioned reagent set, the genome of the recombinant influenza is sub-thread minus strand, segmented RNA, described heavy
The strand RNA of group influenza virus transcribes out the complete positive chain RNA complementary with the strand RNA, and the complete positive chain RNA includes
PB1-RNA, PB2-RNA, PA-RNA, NP-RNA, M-RNA, HA-RNA, NA-RNA and HCKP-RNA；
The PB1-RNA is the RNA of PB1 in encoding influenza virus strain；
The PB2-RNA is the RNA of PB2 in the coding Influenza virus strain；
The PA-RNA is the RNA of PA in the coding Influenza virus strain；
The NP-RNA is the RNA of NP in the coding Influenza virus strain；
The M-RNA is the RNA of M1 and M2 in the coding Influenza virus strain:
The HA-RNA is the RNA of HA in the coding Influenza virus strain；
The NA-RNA is the RNA of NA in the coding Influenza virus strain；
The HCKP-RNA is the RNA for encoding HCKP.
In above-mentioned reagent set, the sequence of the RNA of the coding HCKP can be by the 104-1303 of sequence 2 in sequence table
All T in position replace with U, the constant obtained sequence of other nucleotide.
Wherein, DNA molecular (encoding gene for being named as HCKP) code sequence shown in 104-1303 of sequence 2
HCKP shown in column 1.The sequence of the RNA of the coding HCKP is made of 1200 nucleotide, the sequence of the RNA of the coding HCKP
Column are only that all T in the coding gene sequence of HCKP are replaced with U, the constant obtained sequence of other nucleotide.
In above-mentioned reagent set, the Influenza virus strain can be Influenza virus strain A/PR/8/34.
In above-mentioned reagent set, the genome of the recombinant influenza may include reversed with the RNA of the coding HCKP
The portion gene group of complementary single stranded RNA and Influenza virus strain A/PR/8/34, the genome of the recombinant influenza can also
By the single stranded RNA of the RNA reverse complemental with the coding HCKP and the portion gene group group of Influenza virus strain A/PR/8/34
At；The portion gene group of the Influenza virus strain A/PR/8/34 is by encoding institute in Influenza virus strain A/PR/8/34 genome
State the strand RNA of PB1, the strand RNA of the coding PB2, the strand RNA of the coding PA, the coding NP strand RNA,
Encode the minus strand of the strand RNA of the M1, the strand RNA of the coding M2, the strand RNA of the coding HA and the coding NA
RNA composition.
In above-mentioned reagent set, the expression of the encoding gene of the HCKP can be as shown in 4010-5179 of sequence 2
DNA molecular starting.
Sequence 2 is made of 5200 nucleotide, the sequence of the 104-1303 encoding genes for the HCKP of sequence 2
Column, 4010-5179 of sequence 2 are the sequence for starting the promoter of encoding gene expression of the HCKP.
In order to solve the above technical problems, the present invention also provides the construction methods of recombinant influenza in the reagent set
The construction method of recombinant influenza in the reagent set provided by the present invention, including will contain described in coding
The recombinant vector of the DNA molecular of PB1-RNA, contains coding institute at the recombinant vector containing the DNA molecular for encoding the PB2-RNA
It states the recombinant vector of the DNA molecular of PA-RNA, the recombinant vector containing the DNA molecular for encoding the NP-RNA, contain coding institute
It states the recombinant vector of the DNA molecular of M-RNA, the recombinant vector containing the DNA molecular for encoding the HA-RNA, contain described in coding
It is thin that the recombinant vector of the recombinant vector of the DNA molecular of NA-RNA and the DNA molecular containing the coding HCKP-RNA imports packaging
In born of the same parents, recombinant influenza is obtained.
In the above method, the expression of the DNA molecular of the HCKP-RNA can be as shown in 4010-5179 of sequence 2
DNA molecular starting.
In order to solve the above technical problems, the present invention also provides following any products:
P1, the recombinant influenza；
The genome of P2, the recombinant influenza；
The carrier of P3, encoding gene containing the HCKP；
The expression cassette of P4, encoding gene containing the HCKP；
The encoding gene of P5, the HCKP；
P6、HCKP。
In the said goods, the concretely carrier shown in sequence 2 of carrier described in P3.
In the said goods, the encoding gene of the HCKP can be DNA molecular shown in 104-1303 of sequence 2.
In order to solve the above technical problems, the present invention also provides biomaterials relevant to the recombinant influenza.
Biomaterial relevant to the recombinant influenza provided by the present invention is following E1) appointing into E18)
It is a kind of:
F1 the recombinant vector of the complete DNA molecular) containing the complete positive chain RNA for encoding the recombinant influenza；
E2) complete carrier, by following E2a), E2b), E2c), E2d), E2e), E2f), E2g) and E2h) form:
E2a the recombinant vector) containing the DNA molecular for encoding the PB1-RNA；
E2b the recombinant vector) containing the DNA molecular for encoding the PB2-RNA；
E2c the recombinant vector) containing the DNA molecular for encoding the PA-RNA；
E2d the recombinant vector) containing the DNA molecular for encoding the NP-RNA；
E2e the recombinant vector) containing the DNA molecular for encoding the M-RNA；
E2f the recombinant vector) containing the DNA molecular for encoding the HA-RNA；
E2g the recombinant vector) containing the DNA molecular for encoding the NA-RNA；
E2h the recombinant vector) containing the DNA molecular for encoding the HCKP-RNA；
E3) containing coding E1) recombinant microorganism of the complete DNA molecular；
E4) contain E1) recombinant microorganism of the recombinant vector；
E5) contain E2) recombinant microorganism of the complete carrier；
E6) containing coding E1) the transgenetic animal cell system of the complete DNA molecular；
E7) contain E1) the transgenetic animal cell system of the recombinant vector；
E8) contain E2) the transgenetic animal cell system of the complete carrier；
E9) containing coding E1) the transgenic animals tissue of the complete DNA molecular；
E10) contain E1) the transgenic animals tissue of the recombinant vector；
F11) contain E2) the transgenic animals tissue of the complete carrier；
E12) containing coding E1) transgenic animal organ of the complete DNA molecular；
E13) contain E1) transgenic animal organ of the recombinant vector；
E14) contain E2) transgenic animal organ of the complete carrier；
E15) contain the microorganism of the recombinant influenza；
E16) contain the animal cell line of the recombinant influenza；
E17) contain the animal tissue of the recombinant influenza；
E18) contain the animal organ of the recombinant influenza；
The complete DNA molecular is by encoding the DNA molecular of the PB1-RNA, the DNA molecular of the coding PB2-RNA, compiling
The DNA molecular of the code PA-RNA, the DNA molecular of the coding NP-RNA, the DNA molecular of the coding M-RNA, described in coding
The DNA molecular composition of the DNA molecular of HA-RNA, the DNA molecular of the coding NA-RNA and the coding HCKP-RNA.
In above-mentioned biomaterial, the DNA molecular of the positive chain RNA of the coding HCKP can be 104-1303 of sequence 2
Shown in DNA molecular.
In above-mentioned biomaterial, the transgenetic animal cell system, transgenic animals tissue, the transgenic animals
Organ, the animal cell line, the animal tissue and the animal organ do not include propagation material.
In above-mentioned biomaterial, the recombinant microorganism can be recombinant influenza.
In above-mentioned biomaterial, E15) microorganism, E16) animal cell line, E17) animal tissue and
E18) animal organ can be the host of influenza virus.
In one embodiment of the invention, E2a) recombinant vector is pHW-PB1；E2b) recombinant vector is
pHW-PB2；E2c) recombinant vector is pHW-PA；E2d) recombinant vector is pHW-NP；E2e) recombinant vector is
pHW-M；E2f) recombinant vector is pHW-HA；E2g) recombinant vector is pHW-NA.E2h) recombinant vector is sequence
Carrier pHW-HCKPP-HCKP shown in column 2.In pHW-HCKPP-HCKP, DNA shown in 4010-5179 of sequence 2 points
The expression of gene (i.e. the encoding gene of HCKP), the encoding gene of the HCKP shown in 104-1303 of sub- initiating sequence 2
HCKP shown in coded sequence 1.
In order to solve the above technical problems, the present invention also provides tumors.
The active constituent of tumor provided by the present invention can be the reagent set.
In order to solve the above technical problems, the present invention also provides following any applications:
I, application of the reagent set in preparation tumor；
The application of II, the recombinant influenza in preparation tumor；
The application of III, the genome in preparation tumor；
The application of IV, the biomaterial in preparation tumor；
V, application of the reagent set in treatment tumour；
The application of VI, the recombinant influenza in treatment and/or tumour medicine；
The application of VII, the genome in treatment tumour；
The application of VIII, the biomaterial in treatment tumour.
Heretofore described tumour can be entity tumor, such as liver cancer.The liver cancer concretely primary carcinoma of liver can also be
The liver cancer that HepG2, SMMC-7721, HuH-7 or HuH-7.5 cell line induce.
In the present invention, adriamycin can be Wanle Pharmaceutical Co Ltd, Shenzhen's product, and catalog number is national drug standard
44024359。
It is demonstrated experimentally that reagent set and recombinant influenza FLU-HCKPP-HCKP of the invention can inhibit the life of liver cancer
Life span that is long and can extending animal: the gross tumor volume of FLU-HCKPP-HCKP treatment group is respectively A/PR/8/34 treatment
Organize 0.43 times and 0.41 times with PBS treatment group；The gross tumor volume of reagent set treatment group is respectively DOX group, FLU-HCKPP-
0.73 times, 0.65 times, 0.71 times, 0.26 times of HCKP group, A/PR/8/34+DOX group and PBS group；It is controlled in FLU-HCKPP-HCKP
The time treated when treatment group animal survival rate is 40% away from the 1st time is respectively A/PR/8/34 group and PBS group animal survival rate is
1.30 times and 1.37 times of the time treated when 40% away from the 1st time；Reagent set treatment group nude mice survival rate be 40% when away from
The time of 1st treatment is respectively DOX group, FLU-HCKPP-HCKP group, A/PR/8/34+DOX group and PBS group nude mice survival rate
1.27 times, 1.46 times, 1.46 times, 2 times of the time treated when being 40% away from the 1st time.
It is demonstrated experimentally that recombinant influenza FLU-HCKPP-HCKP of the invention can specifically inhibit the increasing of liver cancer cells
Grow, and the inhibiting effect have recombinant influenza dose dependent: FLU-HCKPP-HCKP to liver cancer cell lines HepG2,
The inhibiting rate of SMMC-7721, HuH-7 and HuH-7.5 are respectively inhibition of the FLU-HCKPP-HCKP to Human normal hepatocyte L-02
5.33,4.14,5.61,4.87 times of rate.
It is demonstrated experimentally that reagent set and recombinant influenza FLU-HCKPP-HCKP of the invention can be used to treat liver
Cancer.
Detailed description of the invention
Fig. 1 is the survival rate of cell after recombinating influenza infection difference cell.
Fig. 2 be different TCID50 multiplicity of infection MOI under the conditions of recombinant influenza to different liver cancer cell lines (HepG2,
HuH-7 influence).Wherein, A is influence of the recombinant influenza to HepG2；B is influence of the recombinant influenza to HuH-7.
Fig. 3 is the treatment results of recombinant influenza and reagent set of the invention to hepatocellular carcinoma in nude mice.
Fig. 4 is the time-to-live that recombinant influenza and reagent set of the invention treat nude mice after nude mice.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The nephrocyte (COS-1) of cercopithecus aethiops SV40 conversion in following embodiments is ATCC (American Type
Culture Collection) cell bank product.Dog kidney cells (MDCK) in following embodiments is ATCC (American
Type Culture Collection) cell bank product.
SPF chicken embryo in following embodiments is Beijing Experimental Amimal Research Centre's product.
Recombinant vector pHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-M, pHW-HA in following embodiments and
PHW-NA (mono- Plasmid system for rapid of Hoffmann E, Mrauss S, Perez D, etal.Eight
Generation of influenza virus vaccines.Vaccine, 2002,20:3165-70) public can be from applicant
Place obtains, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.Recombinant vector
PHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-M, pHW-HA and pHW-NA contain respectively respectively with strains of influenza viruses A/
PR/8/34 (mono- Plasmid system for rapid of Hoffmann E, Mrauss S, Perez D, etal.Fight
Generation of influenza virus vaccines.Vaccine, 2002,20:3165-70) internal viral gene
The DNA fragmentation of skeleton PB1, PB2, PA, NP, M, HA, NA reverse complemental, pHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-
HA and pHW-NA is separately encoded PB1, PB2, PA, NP, HA and NA of strains of influenza viruses A/PR/8/34；PHW-M encoding influenza virus
The M1 and M2 of strain A/PR/8/34.
HepG2, SMMC-7721, HuH-7, HuH-7.5, L-02 in following embodiments are the Chinese People's Liberation Army
Three 02 Hospital's products.
SPF grade nude mice in following embodiments is Beijing Vital River Experimental Animals Technology Co., Ltd.'s product, and article No. is
401.Adriamycin (Doxorubicin, DOX) in following embodiments is Wanle Pharmaceutical Co Ltd, Shenzhen's product, catalogue
Number be national drug standard 44024359.
The rescue of embodiment 1, recombinant influenza
1, the building of recombinant vector
Carrier shown in sequence 2 is named as pHW-HCKPP-HCKP, in pHW-HCKPP-HCKP, the 4010- of sequence 2
Gene shown in 104-1303 of DNA molecular shown in 5179 (DNA molecular is named as HCKPP) initiating sequence 2 (will
The unnamed gene is HCKP gene) expression, which (is named as by protein shown in HCKP gene coded sequence 1
HCKP)。
The DNA molecular as shown in 4010-5200 of sequence 2 is inserted between the BsmB I of carrier pHW2000 identifies sequence
3 ' ends 54-1499 of catenation sequences 2 shown in DNA molecular the obtained DNA moleculars in 5 ' ends, holding carrier pHW2000's
Other sequences are constant, obtain recombinant vector, which is named as p-HCKPP-HCKP.
2, the rescue of recombinant influenza
By the pHW-HCKPP-HCKP of step 1 and recombinant vector pHW-PB1, pHW-PB2, pHW-PA, pHW-NP, pHW-M,
Room temperature in 10 μ L transfection reagents (Effectene, Qiagen Products) is added after each 0.2 μ g mixing of pHW-HA and pHW-NA to make
With 10min, in the nephrocyte (COS-1) and dog kidney cells (MDCK) that cotransfection is converted to cercopithecus aethiops SV40, in 37 DEG C, 5%
CO2Lower culture 60-72 hours, obtains cell suspension, by cell suspension inoculation 9-11 age in days SPF chicken embryo, 37 DEG C of culture 72h, harvest
Chick embryo allantoic liquid simultaneously carries out blood clotting (HA) test and blood clotting inhibition (HI) test according to OIE standard to it.HA and HI test result
It is that positive sample contains the successful recombinant influenza (being named as FLU-HCKPP-HCKP) of rescue, by expanding,
Concentration, purifying obtain FLU-HCKPP-HCKP, and -70 DEG C freeze.
According to the method described above, pHW-HCKPP-HCKP is replaced with into p-HCKPP-HCKP, other steps are constant, carry out weight
The rescue of group influenza virus, does not as a result obtain recombinant influenza.
3, the identification of FLU-HCKPP-HCKP
The susceptible FLU-HCKPP-HCKP of recombined streams transmission electron microscope observing morphology of virus after negative staining that step 2 obtains, as a result
Show that FLU-HCKPP-HCKP meets influenza virus representative configuration feature, there is coating, surface spinosity lug structure, virion size
Between 80-120nm.
By the FLU-HCKPP-HCKP inoculation 9-11 age in days SPF chicken embryo passage of step 2, second generation chick embryo allantoic liquid is taken to extract
Viral RNA amplifies correct PB2, PB1, PA, NP, HA, NA and M genetic fragment of sequence and HCKP base by RT-PCR
Cause.
By the FLU-HCKPP-HCKP of step 2 through chicken embryo mass propgation, be concentrated by ultrafiltration, sucrose gradient centrifugation after purification, into
NP, HA1, HA2, NEP albumen of corresponding size, table can be detected after gel-colored, decoloration in row polyacrylamide gel electrophoresis
The main component of bright antigen is not lost.
Embodiment 2, FLU-HCKPP-HCKP with liver cancer targeting cell and can inhibit the proliferation of liver cancer cells
1, recombinant influenza FLU-HCKPP-HCKP can specifically inhibit the proliferation of liver cancer cells
The FLU-HCKPP-HCKP of 1 step 2 of embodiment is resuspended with PBS, obtains FLU-HCKPP-HCKP suspension.It utilizes
MTS reagent box (Promega) detects recombinant influenza FLU-HCKPP-HCKP to the lethal effect of liver cancer cells, and experiment repeats
Three times, specific step is as follows for repetition experiment every time:
Liver cancer cell lines HepG2, SMMC-7721, HuH-7, HuH-7.5 and Human normal hepatocyte L-02 are inoculated with respectively
In 96 orifice plates containing culture medium, the amount of every hole culture medium is identical, a kind of every cell in hole, two holes of every kind of cell, every hole 104
A cell, in 37 DEG C, 5%CO2After lower culture 24 hours, the multiplicity of infection MOI according to living cells TCID50 is 10, respectively to every
Kind of cell inoculation FLU-HCKPP-HCKP suspension (every kind of cell is inoculated with isometric PBS as control respectively), in 37 DEG C,
5%CO2Under carry out culture 6 days, using MTS reagent box (Promega) detect 490nm under light absorption value, calculate each hole cell
Survival rate, as a result as shown in figure 1 and table 1.
Table 1, FLU-HCKPP-HCKP are to the inhibiting rates of different cells
The results show that suppression of the FLU-HCKPP-HCKP to liver cancer cell lines HepG2, SMMC-7721, HuH-7 and HuH-7.5
Rate processed is respectively FLU-HCKPP-HCKP to 5.33,4.14,5.61,4.87 times of the inhibiting rate of Human normal hepatocyte L-02.Table
Bright, FLU-HCKPP-HCKP can specifically inhibit the proliferation of liver cancer cells.
2, recombinant influenza FLU-HCKPP-HCKP has dose dependent to the inhibiting effect of hepatoma cell proliferation
In triplicate, repeating experiment every time, specific step is as follows for experiment:
Liver cancer cell lines HepG2 is seeded in 96 orifice plates containing culture medium, the amount of every hole culture medium is identical, every hole
104A cell, in 37 DEG C, 5%CO2After lower culture 24 hours, the multiplicity of infection MOI according to living cells TCID50 is 10,5,1 and
0.1 respectively into every hole inoculation step 1 FLU-HCKPP-HCKP suspension, a kind of every multiplicity of infection of living cells TCID50 in hole
MOI, in 37 DEG C, 5%CO2Under carry out culture 6 days, daily using MTS reagent box (Promega) detection 490nm under light absorption value,
Calculate the survival rate (A in Fig. 2) of each hole HepG2 after FLU-HCKPP-HCKP is handled.
According to the method described above, HepG2 is replaced with into HuH-7, keeps other steps constant, obtains FLU-HCKPP-HCKP
The survival rate (B in Fig. 2) of each hole HepG2 after processing.
The results show that recombinant influenza FLU-HCKPP-HCKP is to the inhibiting effect of hepatoma cell proliferation with living cells
The increase of the multiplicity of infection MOI of TCID50 and enhance, show recombinant influenza FLU-HCKPP-HCKP to hepatoma cell proliferation
Inhibiting effect have dose dependent.
3, RT-PCR detects the expression of HCKP
In triplicate, repeating experiment every time, specific step is as follows for experiment:
Liver cancer cell lines HepG2 and HuH-7 and Human normal hepatocyte L-02 are seeded in 96 containing culture medium respectively
In orifice plate, the amount of every hole culture medium is identical, a kind of every cell in hole, two holes of every kind of cell, every hole 104A cell, in 37 DEG C,
5%CO2After lower culture 24 hours, the FLU-HCKPP-HCKP suspension of inoculation step 1 into every hole, every hole 1 × 109Pfu recombination
Influenza virus FLU-HCKPP-HCKP, in 37 DEG C, 5%CO2Lower culture 48 hours, collects the RNA of cell extraction cell, reverse transcription
Afterwards with Primer-1:5 '-CACTTATATT CACCTGCCTCAGGG-3 ' and Primer-2:5 '-CCTAACATAT
CACCTGCCTC G-3 ' is that primer carries out PCR reaction, is made with not being inoculated with HepG2, HuH-7 and L-02 of FLU-HCKPP-HCKP
For negative control.The results show that the HepG2 and HuH-7 of inoculation FLU-HCKPP-HCKP, which are expanded, has arrived size as 1446bp's
DNA fragmentation is inoculated with the L-02 of FLU-HCKPP-HCKP, is not inoculated with HepG2, HuH-7 and L-02 of FLU-HCKPP-HCKP not
It expands to target DNA fragment.Show that recombinant influenza FLU-HCKPP-HCKP is expressed in HepG2 and HuH-7,
It is not expressed in L-02, illustrates the HCKP of the recombinant influenza FLU-HCKPP-HCKP specifically expressing in liver cancer cells.
Embodiment 3, recombinant influenza FLU-HCKPP-HCKP are to the internal inhibiting effect of liver cancer
In triplicate, repeating experiment every time, specific step is as follows for experiment:
The FLU-HCKPP-HCKP of embodiment 1 is resuspended with PBS, obtaining titre is 2 × 109The FLU- of pfu/100 μ l
HCKPP-HCKP suspension；Strains of influenza viruses A/PR/8/34 is resuspended with PBS, obtaining titre is 2 × 109The stream of pfu/100 μ l
Susceptible strain A/PR/8/34 suspension；DOX is dissolved in PBS and obtains DOX solution.
SPF grades of nude mices (weight 18-20g) of 4 week old are taken, every inoculates 5 × 10 in right side oxter6A human liver cancer cell
HuH-7.Tumour major diameter and minor axis, gross tumor volume=(longest diameter × most short diameter are measured twice a week2)/2.Work as gross tumor volume
For 100-150mm3When, nude mice is grouped at random, every group of 5 nude mices, respectively FLU-HCKPP-HCKP group, DOX group,
FLU-HCKPP-HCKP+DOX group, A/PR/8/34 group, A/PR/8/34+DOX group and PBS group.
FLU-HCKPP-HCKP group carries out following treatment: directly to the FLU-HCKPP- of 100 μ l of the intra-tumoral injection of nude mice
First time injection is denoted as treatment the 0th day by HCKP suspension, continuous injection 5 days.
DOX group carries out following treatment: according to the standard of every kg body weight 8mg DOX, respectively in treatment the 0th day and the 3rd day
To nude mice tail vein injection DOX solution.
FLU-HCKPP-HCKP+DOX group carries out following treatment: respectively in treatment the 0th day, the 1st day, the 2nd day, the 3rd day and
The FLU-HCKPP-HCKP suspension of 4th day from the 100 μ l of intra-tumoral injection to nude mice, and according to every kg body weight 8mg DOX's
Standard is treating the 0th day and the 3rd day to nude mice tail vein injection DOX solution respectively.
A/PR/8/34 group carries out following treatment: respectively in treatment the 0th day, the 1st day, the 2nd day, the 3rd day and the 4th day to naked
The A/PR/8/34 suspension of the 100 μ l of intra-tumoral injection of mouse.
A/PR/8/34+DOX group carries out following treatment: respectively in treatment the 0th day, the 1st day, the 2nd day, the 3rd day and the 4th day
To the A/PR/8/34 suspension of the 100 μ l of intra-tumoral injection of nude mice, and according to the standard of every kg body weight 8mg DOX, exist respectively
The 0th day and the 3rd day is treated to nude mice tail vein injection DOX solution.
PBS group carries out following treatment: respectively in the 0th day, the 1st day, the 2nd day, the 3rd day and the 4th day swelling to nude mice for the treatment of
The PBS of 100 μ l of intratumor injection.
Treating the 0th day, the 4th day, the 8th day, the 12nd day, the 16th day, the 20th day, the 24th day, the 28th day, the 32nd respectively
It, the 36th day and the 40th day measurement gross tumor volume, as a result as shown in figure 3 and table 2；Observation mouse is dead in treatment the 0-84 days
Situation is died, counts time of each group nude mice in different survival rates away from first time treatment, as a result as shown in Figure 4.
Table 2, different group gross tumor volume with treatment time variation (mm3)
The results show that in treatment the 40th day, the gross tumor volume of FLU-HCKPP-HCKP group be respectively A/PR/8/34 group with
0.43 times of PBS group and 0.41 times, show that FLU-HCKPP-HCKP can inhibit the growth of liver cancer, FLU-HCKPP-HCKP pairs
Liver cancer has therapeutic effect.In treatment the 40th day, the gross tumor volume of FLU-HCKPP-HCKP+DOX group was respectively DOX group, FLU-
0.73,0.65,0.71,0.26 times of HCKPP-HCKP group, A/PR/8/34+DOX group and PBS group, shows reagent set FLU-
HCKPP-HCKP+DOX can further suppress the growth of liver cancer, and FLU-HCKPP-HCKP+DOX has treatment well to liver cancer
Effect.
The results show that FLU-HCKPP-HCKP can extend the life span of lotus Liver Cancer Bearing Nude Mice: in FLU-HCKPP-HCKP
The time that group nude mice survival rate is treated when being 40% away from the 1st time is respectively A/PR/8/34 group and PBS group nude mice survival rate is 40%
When away from the 1st 1.30 times and 1.37 times of time treated.Reagent set FLU-HCKPP-HCKP+DOX can further extend
The life span of lotus Liver Cancer Bearing Nude Mice: FLU-HCKPP-HCKP+DOX group nude mice survival rate be 40% when away from the 1st time treat when
Between be respectively DOX group, FLU-HCKPP-HCKP group, A/PR/8/34+DOX group and PBS group nude mice survival rate be 40% when away from the 1st
1.27 times, 1.46 times, 1.46 times, 2 times of the time of secondary treatment.
Claims (4)
1. treating the reagent set of liver cancer, it is made of adriamycin and recombinant influenza；The recombinant influenza expresses albumen
Matter, the amino acid sequence of the protein is as shown in sequence 1 in sequence table；The genome of the recombinant influenza is negative for sub-thread
Chain, segmented RNA, the strand RNA of the recombinant influenza transcribe out the complete positive chain RNA complementary with the strand RNA,
The complete positive chain RNA includes PB1-RNA, PB2-RNA, PA-RNA, NP-RNA, M-RNA, HA-RNA, NA-RNA and HCKP-
RNA；
The PB1-RNA is the RNA of PB1 in encoding influenza virus strain；
The PB2-RNA is the RNA of PB2 in the coding Influenza virus strain；
The PA-RNA is the RNA of PA in the coding Influenza virus strain；
The NP-RNA is the RNA of NP in the coding Influenza virus strain；
The M-RNA is the RNA of M1 and M2 in the coding Influenza virus strain；
The HA-RNA is the RNA of HA in the coding Influenza virus strain；
The NA-RNA is the RNA of NA in the coding Influenza virus strain；
The HCKP-RNA is the RNA of code for said proteins.
2. reagent set according to claim 1, it is characterised in that: the sequence of the RNA of the code for said proteins is
All T in 104-1303 of sequence 2 in sequence table are replaced with into U, the constant obtained sequence of other nucleotide；Institute
Stating Influenza virus strain is Influenza virus strain A/PR/8/34.
3. treating liver-cancer medicine, active constituent is reagent set of any of claims 1 or 2.
4. application of the reagent set of any of claims 1 or 2 in preparation treatment liver-cancer medicine.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810961A (en) * | 2006-02-22 | 2006-08-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Recombinant influenza virus and its prepn and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015063085A1 (en) * | 2013-10-28 | 2015-05-07 | Thomas Muster | Novel influenza virus vector for virotherapy |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1810961A (en) * | 2006-02-22 | 2006-08-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Recombinant influenza virus and its prepn and application |
Non-Patent Citations (2)
| Title |
|---|
| "Generation of Replication-Competent Recombinant Influenza A Viruses";Feng Li et al.,;《JOURNAL OF VIROLOGY》;20101231;第84卷(第22期);第12075-12081页 * |
| "甲型H1N1 流感重组病毒模型的建立及应用";吴彦霖;《药物分析杂志》;20161231;第36卷(第7期);第1162-1168页 * |
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