CN106318915B - Recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP and diagnostic kit - Google Patents
Recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP and diagnostic kit Download PDFInfo
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Abstract
The invention discloses a recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP and a corresponding diagnostic kit thereof, wherein a DNA fragment with the length of 25bp is inserted between a telomerase reverse transcriptase promoter and an ICP4 gene on a genome of the virus, a GFP expression box is inserted into an ICP34.5 site, and the sequence of the DNA fragment is AGCAGGACCTCGCCCTTGGGACGAC. The virus has high-titer reproductive capacity selectively, and the virus has stable propagation titer at 10 in lung cancer cells6The above. The invention uses a synthesized random nucleotide short sequence to carry out site-directed recombination with HSV1-hTERTp _ ICP4 virus genome (between hTERTp and ICP 4). The change of the short sequence can not only increase the propagation titer of the virus, but also lead different viruses to selectively propagate with high titer against different types of tumor cells. Some viruses propagate better in lung cancer cells, and some viruses generate higher titer in liver cancer cells, and the like.
Description
Technical Field
The invention relates to a recombinant herpes simplex virus, in particular to a recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP and a corresponding diagnostic kit thereof.
Background
Telomeres are a special structure at the end of chromosomes in eukaryotes, which serves to maintain stable chromosome structure, including preventing fusion of chromosome ends, protecting chromosome structural genes and avoiding loss of genetic information during replication. Telomerase is a reverse transcriptase consisting of small-molecule RNA and protein, and can synthesize telomere DNA by using self RNA as a template to make up telomeres which are gradually shortened along with mitosis of cells. It has three main components: human telomerase RNA (hTR), telomerase-associated protein (TP 1/TLP1), human telomerase reverse transcriptase (hTERT). Telomerase RNA is expressed in most cells, while human telomerase reverse transcriptase is the rate-limiting component of telomerase, is expressed only in telomerase positive cells, and is related to telomerase activity. Although some tumor cells maintain telomere length through an Alternative Lengthening of Telomeres (ALT) mechanism upon recombination, more than 90% of tumor cells activate telomerase by up-regulating hTERT, and hTERT is expressed only in a very few normal somatic cells. The telomerase promoter (hTERTp) is known to start a transcription process only in cells with high telomerase activity, which provides a good opportunity for tumor targeted therapy.
Infected Cell Proteins (ICPs) expressed by Herpes Simplex Virus (HSV) are classified into three grades: initial (IE), early (early), and late (late). Wherein the IE protein is a key protein affecting viral replication, including: ICP0, ICP4, ICP22, ICP27 and ICP47, of which ICP4 is the most critical replication-related protein. If the intrinsic promoter of the ICP4 gene is replaced with hTERTp, it is possible to selectively propagate HSV in tumor cells.
A human telomerase reverse transcriptase promoter (hTERTP) is artificially synthesized to replace an ICP4 original promoter in the genome of the existing type I oncolytic herpes simplex virus (17+ strain) in a laboratory, so that a novel virus HSV-hTERTP _ ICP4 is constructed. The recombinant virus can selectively grow and propagate in human tumor cells, but not in human normal cells.
The construction of plasmid containing hTERTp _ ICP4 expression cassette is the basis of the constructed HSV-hTERTp _ ICP4 virus, and the original HSV-hTERTp _ ICP4 is constructed by using the plasmid containing the hTERTp _ ICP4 expression cassette, and the recombinant virus can be selectively propagated in tumor cells, but has lower propagation titer (10)4/ml)。
In order to overcome the defect of low propagation titer, the construction of a virus with high propagation titer is urgently needed.
Disclosure of Invention
The invention aims to solve the defect of low virus propagation titer in the prior art, and provides a recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP, wherein the virus has high selective titer fertility, and the virus propagation titer in lung cancer cells is stable at 106The above.
The invention also provides a diagnostic kit prepared from the recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP.
In order to achieve the purpose, the recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP provided by the invention is characterized in that a DNA fragment with the length of 25bp is inserted between a telomerase reverse transcriptase promoter and an ICP4 gene on a genome of the virus, and a GFP expression cassette is inserted into an ICP34.5 site, wherein the sequence of the DNA fragment is AGCAGGACCTCGCCCTTGGGACGAC.
The invention also provides a preparation method of the recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP, which comprises the following steps:
1) replacing an ICP4 gene promoter in the herpes simplex virus containing the ICP4 gene with a human telomerase reverse transcriptase promoter hTERTp to construct the recombinant herpes simplex virus HSV-hTERTp _ ICP 4:
(1) construction of shuttle plasmids pICP4del-hTERTp _ ICP4 and pICP4del-eGFP
a. Culturing a herpes simplex virus containing an ICP4 gene by using a BHK cell, and purifying a genome DNA of the herpes simplex virus;
b. amplification of the flanking sequences upstream of the ICP4 gene: using the virus genome DNA obtained in the step a) as a template, and using the following ICP4USf forward primer and ICP4USr reverse primer:
ICP4USf forward primer: ccctccagacgcaccggagtcggggg the flow of the air in the air conditioner,
ICP4USr reverse primer: aagtcgactctagaggatcgatctctgacctgagattggcggcactgaggta
Amplifying the upstream flanking sequence of the ICP4 gene;
c. amplification of flanking sequences downstream of the ICP4 gene: and (b) taking the virus genome DNA obtained in the step a as a template, and using the following ICP4DSf forward primer and ICP4USr reverse primer:
ICP4DSf forward primer: aaaagtcgacctgcaggcatgctaacgaggaacgggcagggggc
ICP4DSr reverse primer: aaaaaagcttgcatgcccacgtgcgcggggccagacgggct
Amplifying sequences flanking the ICP4 gene downstream;
the upstream and downstream flanking sequences were cloned into the pSP73 plasmid, and pICP4del-eGFP plasmids were constructed: mixing the SalI digested upstream flanking sequence of the amplified ICP4 gene and the SalI/HindIII double digested downstream flanking sequence of the ICP4 gene and connecting the mixture to the EcoRV/HindIII site of pSP73 to obtain pICP4 del; cutting an eGFP expression cassette controlled by a CMV promoter from pcDNA3.1-eGFP by EcoRI/XhoI, filling the tail end by T4DNA polymerase, and inserting the cut end into an EcoRV site of pICP4del to obtain pICP4 del-eGFP;
d. three sequences in the ICP4 gene are amplified by three times of PCR:
first, the following primers were used:
ICP4-1st forward primer: ttttttgaattcatggcgtcggagaacaagcagcgcc
ICP4-1st reverse primer: tggagccaccccatggcctccgcgt
ICP4-2nd forward primer: cgacgccgcgcagcagtacgccctg
ICP4-2nd reverse primer: cggcgggggcgggcccggcgcaccg
ICP4-3rd forward primer: cctcatgtttgacccgcgggccctg
ICP4-3rd reverse primer: ttttttctcgagttacagcaccccgtccccctcgaac
Using the virus genome DNA obtained in the step a) as a template, respectively amplifying three segments of gene fragments ICP4-1st, ICP4-2nd and ICP4-3rd, and then respectively inserting the three segments of gene fragments into the EcoRV site of the pSP73 plasmid to construct the following three plasmids: pSP73-ICP4-1st, pSP73-ICP4-2nd and pSP73-ICP4-3rd, wherein EcoRI and BsrGI are used for cutting ICP4-1st, BsrGI and PvuI are used for cutting ICP4-2nd and PvuI and XhoI are used for cutting ICP-3rd for standby;
e. cutting an hTERTp fragment from a plasmid containing a human telomerase reverse transcriptase promoter hTERTp by using NruI and HindIII, and replacing a CMV promoter cut from pcDNA3-NHN by using NruI and HindIII to obtain a plasmid pcDNA3-NHN-hTERTp, wherein the pcDNA3-NHN is obtained by inserting a NheI-HapI-NheI enzyme cutting site sequence into the NheI site of the pcDNA 3;
f. mixing ICP4-1st, ICP4-2nd and ICP4-3rd obtained in the step c) and connecting to EcoRI and XhoI sites of pcDNA3-NHN-hTERTp obtained in the step d) to obtain plasmid pcDNA3-NHN-hTERTp _ ICP 4;
g. digesting the plasmid pICP4del containing the upstream and downstream flanking sequences of the ICP4 gene obtained in the step b) by SalI, filling the ends of the plasmid pICP4del, using PmeI and HpaI to cut out an hTERTp _ ICP4 expression cassette fragment from the plasmid pcDNA3-NHN-hTERTp _ ICP4 obtained in the step e), and connecting the fragment with the digested and standby pICP4del plasmid to construct a plasmid pICP4del-hTERTp _ ICP 4;
h. construction of BHK-ICP4 helper cells: cutting the ICP4 gene from the plasmid pcDNA3-NHN-hTERTp _ ICP4 in the step e) by EcoRI and XhoI, and cloning to the EcoRI site and the XhoI site at the downstream of a CMV promoter in the pcDNA3 to obtain a pcDNA3-CMV-ICP4 plasmid; transfecting the plasmid pcDNA3-CMV-ICP4 into BHK cells, recombining the plasmid DNA pcDNA3-CMV-ICP4 into a genome of the BHK cells to ensure that some BHK recombined cells obtain resistance to neomycin and express ICP4, killing the non-recombined BHK cells by using antibiotic G418, performing subclone screening for several rounds, and screening BHK-ICP4 helper cells expressing ICP4 by using an RT-PCR method;
(2) knocking out an ICP4 gene promoter in a genome and inserting a granzyme reverse transcriptase promoter hTERTp promoter:
a. culturing the herpes simplex virus containing the ICP4 gene by using BHK cells, and extracting virus genome DNA;
b. co-transferring the virus genome DNA in the step A and the plasmid pICP4del-eGFP obtained in the step b) of the step 1) into the BHK-ICP4 auxiliary cell obtained in the step 1), and replacing an ICP4 gene of the herpes simplex virus HSV containing the ICP4 gene by a green fluorescent protein GFP expression cassette in the plasmid pICP4del-eGFP through homologous recombination, so that the virus plaques of the recombinant virus emit green fluorescence, and selecting green fluorescent virus plaques through a plurality of cycles of plaque purification, thereby purifying the recombinant virus HSV-d4 GFP;
c. culturing HSV-d4GFP virus and extracting genome DNA;
d. co-transferring the genomic DNA of the recombinant virus HSV-d4GFP and the DNA of the plasmid pICP4del-hTERTp _ ICP4 obtained in the f small step in the step 1) into BHK-ICP4 auxiliary cells, and replacing a green fluorescent protein GFP expression cassette of the recombinant virus HSV-d4GFP by an hTERTp _ ICP4 expression cassette through homologous recombination, so that the virus spots of the new recombinant virus do not emit green fluorescence, selecting non-fluorescent virus spots through a plurality of rounds of plaque purification, and purifying the recombinant herpes simplex virus HSV-hTERTp _ ICP 4;
2) construction of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ Lungca
(1) Complementary 145-base-long DNA fragments, respectively, hp-ICP4 positive and hp-ICP4 negative, were synthesized as follows. Wherein the N sequence (25 bases) is a random base, one side of the flanking known sequences is homologous with the 3 'terminal sequence of the telomerase reverse transcriptase promoter at the HSV-hTERTp _ ICP4, and the other side of the flanking known sequences is homologous with the 5' terminal sequence of the ICP4 gene;
hp-ICP4 positive:
CCGCGAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNGAATTCATGGCGTCGGAGAACAAGCAGCGCCCCGGCTCCCCGGGCCCCACCGACGGGCCG
hp-ICP4 negative:
CGGCCCGTCGGTGGGGCCCGGGGAGCCGGGGCGCTGCTTGTTCTCCGACGCCATGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTCGCGG
(2) constructing a recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA:
extracting HSV-hTERTp _ ICP4 virus genome DNA, carrying out positive and negative homologous recombination on the synthesized DNA fragment hp-ICP4 and hp-ICP4, and screening by using lung cancer cells A549 to obtain HSV-hTERTp _ ICP4_ LungCA (HSV _ LungCA for short) with high titer aiming at the lung cancer cells;
(3) culturing and purifying HSV _ LungCA recombinant virus, extracting virus genome DNA, sequencing the combination part of a telomerase reverse transcriptase promoter and an ICP4 gene, and determining that the sequence of 25 bases is AGCAGGACCTCGCCCTTGGGACGAC;
3) construction of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP
(1) Construction of shuttle plasmid pdICP34.5-eGFP
a. Culturing HSV-hTERTp _ ICP4 by using BHK-ICP4 cells, and purifying the genomic DNA of the HSV-hTERTp _ ICP 4;
b. amplifying the flanking sequence of the ICP34.5 gene upstream: taking the virus genome DNA obtained in the step a as a template, and using the following ICP34.5USf forward primer and ICP34.5USr reverse primer:
ICP34.5USf：CTCTGACCTGAGATTGGCGGCACTG
ICP34.5USr：
GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGCTCCTGACCGCGGG
amplifying an upstream flanking sequence of the ICP34.5 gene;
c. amplification of flanking sequences downstream of the ICP34.5 gene: using the virus genome DNA obtained in the step a) as a template, and using the following ICP34.5DSf forward primer and ICP34.5DSr reverse primer:
ICP34.5DSf：
GCGGCCGCAGCGCTGCGGCCGCCAGCGCGGCGGGGCCCGGCCAACCA
ICP34.5DSr：TTCTTCCCTCTTCTCCCGCCCTCCA
amplifying the downstream flanking sequence of the ICP34.5 gene;
d. connecting the upstream and downstream flanking sequences of the ICP34.5 gene: taking the ICP34.5 upstream and downstream flanking sequences obtained in the steps b) and c) as templates, and amplifying and connecting the ICP34.5 gene upstream and downstream flanking sequences by using ICP34.5USf and ICP34.5DSr;
e. cloning the upstream and downstream flanking sequences onto pSP72 plasmid to construct pdICP34.5;
f. obtaining an eGFP expression sequence from pcDNA3.1-eGFP and inserting the eGFP expression sequence into an AfeI site of pdICP34.5 to obtain pdICP34.5-eGFP;
(2) construction of HSV-hTERTp _ ICP4_ LungCA-GFP
A. Culturing HSV-hTERTp _ ICP4_ LungCA (HSV _ LungCA) by using A549 cells, and purifying the genomic DNA of the HSV-hTERTp _ ICP4_ LungCA;
B. and (2) co-transferring the virus genome DNA in the step (A) and the plasmid pdICP34.5-eGFP in the step (1) into a lung cancer A549 cell, replacing an ICP34.5 gene of herpes simplex virus HSV _ LungCA containing the ICP34.5 gene with a green fluorescent protein GFP expression cassette in the plasmid pdICP34.5-eGFP through homologous recombination, enabling virus plaques of the recombinant virus to emit green fluorescence, carrying out plaque purification for several times, selecting the green fluorescent virus plaques, and purifying the recombinant virus HSV-hTERTP _ 4_ LungCA-GFP (HSV _ LungCA-GFP for short).
The invention also provides application of the recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP in preparation of a tumor diagnostic kit.
The diagnostic kit consists of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP, human CD45 antibody marked by PEcy5 and erythrocyte lysate.
Further, the content of the herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP is 5E6-5E7CCID50/ml, and the content of the human CD45 antibody marked by the PEcy5 is 0.1-0.5 mg/ml.
Still further, the erythrocyte lysate contains ammonium chloride, disodium ethylene diamine tetraacetate, potassium bicarbonate and ultrapure water, wherein the content of the ammonium chloride is 0.10-0.20M, the content of the disodium ethylene diamine tetraacetate is 0.5-2 mM, and the content of the potassium bicarbonate is 5-15 mM. Preferably, the content of ammonium chloride is 0.15M, the content of disodium ethylenediaminetetraacetate is 1mM, and the content of potassium bicarbonate is 10 mM.
Theoretical basis of the invention
The base sequence composition between the promoter and the regulatory gene thereof may influence the expression amount of the gene, and the expression amount of the virus early protein ICP4 may influence the reproductive capacity and titer of the virus in cells. Based on HSV-hTERTp _ ICP4, a synthetic random nucleotide short sequence (25nt) is inserted between hTERTp and ICP4 of a virus genome in a fixed-point mode by using a homologous recombination method, and different types of tumor cells are used for screening and propagating recombinant viruses with high titer. We have surprisingly found that sequence variations between the promoter (hTERTp) and the gene (ICP4) lead to large differences in the reproductive titers of different recombinant viruses in different types of tumor cells.
The invention has the beneficial effects that:
the invention uses a synthesized random nucleotide short sequence to carry out site-directed recombination with HSV1-hTERTp _ ICP4 virus genome (between hTERTp and ICP 4). The change of the short sequence can not only increase the propagation titer of the virus, but also lead different viruses to selectively propagate with high titer against different types of tumor cells. Some viruses propagate better in lung cancer cells, and some viruses generate higher titer in liver cancer cells, and the like.
Drawings
FIG. 1 is a comparison of virus-infected cells;
in the figure, FIG. 1A is a comparison graph of virus-infected A549 cells;
FIG. 1B is a comparison of virus-infected PG cells;
FIG. 1C is a comparison of virus-infected NCI-H1755 cells;
fig. 2 is a CTC detection map of non-small cell lung cancer.
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples.
Example 1
A preparation method of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP comprises the following steps:
1) replacing an ICP4 gene promoter in the herpes simplex virus containing the ICP4 gene with a human telomerase reverse transcriptase promoter hTERTp to construct the recombinant herpes simplex virus HSV-hTERTp _ ICP 4:
(1) construction of shuttle plasmids pICP4del-hTERTp _ ICP4 and pICP4del-eGFP
a. Culturing a herpes simplex virus containing an ICP4 gene by using a BHK cell, and purifying a genome DNA of the herpes simplex virus;
b. amplification of the flanking sequences upstream of the ICP4 gene: using the virus genome DNA obtained in the step a) as a template, and using the following ICP4USf forward primer and ICP4USr reverse primer:
ICP4USf forward primer: ccctccagacgcaccggagtcggggg the flow of the air in the air conditioner,
ICP4USr reverse primer: aagtcgactctagaggatcgatctctgacctgagattggcggcactgaggta
Amplifying the upstream flanking sequence of the ICP4 gene;
c. amplification of flanking sequences downstream of the ICP4 gene: and (b) taking the virus genome DNA obtained in the step a as a template, and using the following ICP4DSf forward primer and ICP4USr reverse primer:
ICP4DSf forward primer: aaaagtcgacctgcaggcatgctaacgaggaacgggcagggggc
ICP4DSr reverse primer: aaaaaagcttgcatgcccacgtgcgcggggccagacgggct
Amplifying sequences flanking the ICP4 gene downstream;
the upstream and downstream flanking sequences were cloned into the pSP73 plasmid, and pICP4del-eGFP plasmids were constructed: mixing the SalI digested upstream flanking sequence of the amplified ICP4 gene and the SalI/HindIII double digested downstream flanking sequence of the ICP4 gene and connecting the mixture to the EcoRV/HindIII site of pSP73 to obtain pICP4 del; cutting an eGFP expression cassette controlled by a CMV promoter from pcDNA3.1-eGFP by EcoRI/XhoI, filling the tail end by T4DNA polymerase, and inserting the cut end into an EcoRV site of pICP4del to obtain pICP4 del-eGFP;
d. three sequences in the ICP4 gene are amplified by three times of PCR:
first, the following primers were used:
ICP4-1st forward primer: ttttttgaattcatggcgtcggagaacaagcagcgcc
ICP4-1st reverse primer: tggagccaccccatggcctccgcgt
ICP4-2nd forward primer: cgacgccgcgcagcagtacgccctg
ICP4-2nd reverse primer: cggcgggggcgggcccggcgcaccg
ICP4-3rd forward primer: cctcatgtttgacccgcgggccctg
ICP4-3rd reverse primer: ttttttctcgagttacagcaccccgtccccctcgaac
Using the virus genome DNA obtained in the step a) as a template, respectively amplifying three segments of gene fragments ICP4-1st, ICP4-2nd and ICP4-3rd, and then respectively inserting the three segments of gene fragments into the EcoRV site of the pSP73 plasmid to construct the following three plasmids: pSP73-ICP4-1st, pSP73-ICP4-2nd and pSP73-ICP4-3rd, wherein EcoRI and BsrGI are used for cutting ICP4-1st, BsrGI and PvuI are used for cutting ICP4-2nd and PvuI and XhoI are used for cutting ICP-3rd for standby;
e. cutting an hTERTp fragment from a plasmid containing a human telomerase reverse transcriptase promoter hTERTp by using NruI and HindIII, and replacing a CMV promoter cut from pcDNA3-NHN by using NruI and HindIII to obtain a plasmid pcDNA3-NHN-hTERTp, wherein the pcDNA3-NHN is obtained by inserting a NheI-HapI-NheI enzyme cutting site sequence into the NheI site of the pcDNA 3;
f. mixing ICP4-1st, ICP4-2nd and ICP4-3rd obtained in the step c) and connecting to EcoRI and XhoI sites of pcDNA3-NHN-hTERTp obtained in the step d) to obtain plasmid pcDNA3-NHN-hTERTp _ ICP 4;
g. digesting the plasmid pICP4del containing the upstream and downstream flanking sequences of the ICP4 gene obtained in the step b) by SalI, filling the ends of the plasmid pICP4del, using PmeI and HpaI to cut out an hTERTp _ ICP4 expression cassette fragment from the plasmid pcDNA3-NHN-hTERTp _ ICP4 obtained in the step e), and connecting the fragment with the digested and standby pICP4del plasmid to construct a plasmid pICP4del-hTERTp _ ICP 4;
h. construction of BHK-ICP4 helper cells: cutting the ICP4 gene from the plasmid pcDNA3-NHN-hTERTp _ ICP4 in the step e) by EcoRI and XhoI, and cloning to the EcoRI site and the XhoI site at the downstream of a CMV promoter in the pcDNA3 to obtain a pcDNA3-CMV-ICP4 plasmid; transfecting the plasmid pcDNA3-CMV-ICP4 into BHK cells, recombining the plasmid DNA pcDNA3-CMV-ICP4 into a genome of the BHK cells to ensure that some BHK recombined cells obtain resistance to neomycin and express ICP4, killing the non-recombined BHK cells by using antibiotic G418, performing subclone screening for several rounds, and screening BHK-ICP4 helper cells expressing ICP4 by using an RT-PCR method;
(2) knocking out an ICP4 gene promoter in a genome and inserting a granzyme reverse transcriptase promoter hTERTp promoter:
a. culturing the herpes simplex virus containing the ICP4 gene by using BHK cells, and extracting virus genome DNA;
b. co-transferring the virus genome DNA in the step A and the plasmid pICP4del-eGFP obtained in the step b) of the step 1) into the BHK-ICP4 auxiliary cell obtained in the step 1), and replacing an ICP4 gene of the herpes simplex virus HSV containing the ICP4 gene by a green fluorescent protein GFP expression cassette in the plasmid pICP4del-eGFP through homologous recombination, so that the virus plaques of the recombinant virus emit green fluorescence, and selecting green fluorescent virus plaques through a plurality of cycles of plaque purification, and purifying the recombinant virus HSV-d4 GFP;
c. culturing HSV-d4GFP virus and extracting genome DNA;
d. co-transferring the genomic DNA of the recombinant virus HSV-d4GFP and the DNA of the plasmid pICP4del-hTERTp _ ICP4 obtained in the f small step in the step 1) into BHK-ICP4 auxiliary cells, and replacing a green fluorescent protein GFP expression cassette of the recombinant virus HSV-d4GFP by an hTERTp _ ICP4 expression cassette through homologous recombination, so that the virus spots of the new recombinant virus do not emit green fluorescence, selecting non-fluorescent virus spots through a plurality of rounds of plaque purification, and purifying the recombinant herpes simplex virus HSV-hTERTp _ ICP 4;
2) construction of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ Lungca
(1) Complementary 145-base-long DNA fragments, respectively, hp-ICP4 positive and hp-ICP4 negative, were synthesized as follows. Wherein the N sequence (25 bases) is a random base, one side of the flanking known sequences is homologous with the 3 'terminal sequence of the telomerase reverse transcriptase promoter at the HSV-hTERTp _ ICP4, and the other side of the flanking known sequences is homologous with the 5' terminal sequence of the ICP4 gene;
hp-ICP4 positive:
CCGCGAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNGAATTCATGGCGTCGGAGAACAAGCAGCGCCCCGGCTCCCCGGGCCCCACCGACGGGCCG
hp-ICP4 negative:
CGGCCCGTCGGTGGGGCCCGGGGAGCCGGGGCGCTGCTTGTTCTCCGACGCCATGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTCGCGG
(2) constructing a recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA:
extracting HSV-hTERTp _ ICP4 virus genome DNA, carrying out positive and negative homologous recombination on the synthesized DNA fragment hp-ICP4 and hp-ICP4, and screening by using lung cancer cells A549 to obtain HSV-hTERTp _ ICP4_ LungCA (HSV _ LungCA for short) with high titer aiming at the lung cancer cells;
(3) culturing and purifying HSV _ LungCA recombinant virus, extracting virus genome DNA, sequencing the combination part of a telomerase reverse transcriptase promoter and an ICP4 gene, and determining that the sequence of 25 bases is AGCAGGACCTCGCCCTTGGGACGAC;
3) construction of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP
(1) Construction of shuttle plasmid pdICP34.5-eGFP
a. Culturing HSV-hTERTp _ ICP4 by using BHK-ICP4 cells, and purifying the genomic DNA of the HSV-hTERTp _ ICP 4;
b. amplifying the flanking sequence of the ICP34.5 gene upstream: taking the virus genome DNA obtained in the step a as a template, and using the following ICP34.5USf forward primer and ICP34.5USr reverse primer:
ICP34.5USf：CTCTGACCTGAGATTGGCGGCACTG
ICP34.5USr：
GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGCTCCTGACCGCGGG
amplifying an upstream flanking sequence of the ICP34.5 gene;
c. amplification of flanking sequences downstream of the ICP34.5 gene: using the virus genome DNA obtained in the step a) as a template, and using the following ICP34.5DSf forward primer and ICP34.5DSr reverse primer:
ICP34.5DSf：
GCGGCCGCAGCGCTGCGGCCGCCAGCGCGGCGGGGCCCGGCCAACCA
ICP34.5DSr：TTCTTCCCTCTTCTCCCGCCCTCCA
amplifying the downstream flanking sequence of the ICP34.5 gene;
d. connecting the upstream and downstream flanking sequences of the ICP34.5 gene: taking the ICP34.5 upstream and downstream flanking sequences obtained in the steps b) and c) as templates, and amplifying and connecting the ICP34.5 gene upstream and downstream flanking sequences by using ICP34.5USf and ICP34.5DSr;
e. cloning the upstream and downstream flanking sequences onto pSP72 plasmid to construct pdICP34.5;
f. obtaining an eGFP expression sequence from pcDNA3.1-eGFP and inserting the eGFP expression sequence into an AfeI site of pdICP34.5 to obtain pdICP34.5-eGFP;
(2) construction of HSV-hTERTp _ ICP4_ LungCA-GFP
A. Culturing HSV-hTERTp _ ICP4_ LungCA (HSV _ LungCA) by using A549 cells, and purifying the genomic DNA of the HSV-hTERTp _ ICP4_ LungCA;
B. and (2) co-transferring the virus genome DNA in the step (A) and the plasmid pdICP34.5-eGFP in the step (1) into a lung cancer A549 cell, replacing an ICP34.5 gene of herpes simplex virus HSV _ LungCA containing the ICP34.5 gene with a green fluorescent protein GFP expression cassette in the plasmid pdICP34.5-eGFP through homologous recombination, enabling virus plaques of the recombinant virus to emit green fluorescence, carrying out plaque purification for several times, selecting the green fluorescent virus plaques, and purifying the recombinant virus HSV-hTERTP _ 4_ LungCA-GFP (HSV _ LungCA-GFP for short).
Example 2 HSV _ LungCA-GFP selectively proliferates in lung cancer cells and produces high titer virus:
lung cancer cells (A549 and PG), liver cancer cells (HepG2 and HuH7), prostate cancer cells PC-3, stomach cancer cells BGC823, colon cancer cells HT-29, human erythroleukemia cells TF-1, lymphoma cells U937, breast cancer cells MD-MB-231, pharyngeal squamous carcinoma cells Fadu and melanoma cells A375 were infected with HSV _ LungCA-GFP at MOI of 0.01, and virus titers were measured at 6, 12, 24, 48 and 72 hours. The results are shown in Table 1. Experimental data show that the lung cancer cell infection effect of HSV _ LungCA-GFP is best, and the virus propagation titer is obviously higher (more than 2 log) than that of other cancer cell propagation viruses after 24 hours or later.
Example 3 HSV _ LungCA-GFP produces higher titers of virus in lung cancer cells than HSV _ ICP4
Lung cancer cells a549, PG, and NCI-H1755 were infected with HSV _ LungCA and HSV _ ICP4 at MOI of 0.01, respectively, and virus titers were detected at 6, 12, 24, 48, and 72 hours. The results are shown in FIGS. 1-2. Experimental data show that HSV _ Lungca infects 3 kinds of lung cancer cells, and the virus propagation titer reaches 10 after 24 hours or later6Above, HSV _ ICP4 has low virus titer and peak value of only 10 although it can be propagated in 3 lung cancer cells4. The two viruses infected lung cancer cells, whose reproductive titers differed by 2 logs.
Example 4 detection of Circulating Tumor Cells (CTC) in blood of Lung cancer patient by tumor detection kit
The experiment is divided into two groups, 50 lung cancer patients and 50 normal volunteers. Each tested person is detected according to the following steps:
1) collecting peripheral venous blood 4ml, and centrifuging at 1800r/min (rotating speed of 500g according to different models of centrifuges in adjustment period) for 5 min;
2) removing upper plasma layer (avoiding destroying leukocyte layer, and retaining part of plasma), and transferring collected cells into 50ml centrifuge tube;
3) adding 24ml of erythrocyte lysate, gently mixing, standing at room temperature for 5-10 min until the solution becomes transparent, and centrifuging at 1800r/min for 5 min;
4) removing supernatant, adding 5ml PBS, gently blowing and mixing with a pipette gun, and centrifuging at 1800r/min for 5 min;
5) removing supernatant, adding 2ml DMEM to suspend the cells, mixing uniformly, and spreading 1ml of the suspension to 2 holes of a cell culture plate;
6) add 100. mu.l HSV _ LungCA-GFP into each well, gently shake the cell culture plate to mix all reagents well, place at 37 ℃ with 5% CO2Incubating for 1 h;
7) after incubation was complete, 1ml of DMEM complete medium (DMEM complete medium configuration: mixing FBS and DMEM medium at a ratio of 1: 9), gently shaking the cell culture plate, and continuing to perform cell culture at 37 deg.C and 5% CO2Incubating for 20-24 h;
8) transferring the culture samples of 2 sample holes of the same sample into 1 incubation tube, and centrifuging at 1800r/min for 5 min;
9) removing supernatant, adding 4ml PBS, gently blowing and mixing with a pipette gun, and centrifuging at 1800r/min for 5 min;
10) removing supernatant, adding 800. mu.l PBS to resuspend cells, mixing uniformly, then adding 200. mu.l PEcy5 labeled human CD45 antibody under the condition of keeping out of the sun, mixing uniformly, and incubating for 30min at room temperature in the absence of the light (the PEcy5 fluorescence signal is easy to quench under the condition of visible light, so that the preservation and reaction in the absence of the light are ensured if necessary);
11) after the incubation is finished, 3ml PBS is added, mixed evenly and centrifuged for 5min at 1500 r/min. The supernatant was removed, resuspended in 150. mu.l PBS, mixed well, aspirated and counted by flow cytometry for PEcy5 negative and green fluorescent cells. The positive result judgment standard of the method is more than or equal to 4 green fluorescent cells/4 ml blood.
As shown in fig. 2: CTC (green fluorescent cells) detected in 4ml of blood of a patient with lung cancer are all more than 6 (the median value is 10); CTC cells were not detected in the vast majority of 4ml blood from the healthy volunteer control group, nor did the number of cells detected in the blood of a few healthy volunteers exceed the threshold.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (4)
1. A preparation method of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP is characterized by comprising the following steps: the method comprises the following steps:
1) replacing an ICP4 gene promoter in the herpes simplex virus containing the ICP4 gene with a human telomerase reverse transcriptase promoter hTERTp to construct a recombinant herpes simplex virus HSV-hTERTp _ ICP 4:
(1) construction of shuttle plasmids pICP4del-hTERTp _ ICP4 and pICP4del-eGFP
a. Culturing a herpes simplex virus containing an ICP4 gene by using a BHK cell, and purifying a genome DNA of the herpes simplex virus; b. amplification of the flanking sequences upstream of the ICP4 gene: and (b) taking the virus genome DNA obtained in the step a as a template, and using the following ICP4USf forward primer and ICP4USr reverse primer:
ICP4USf forward primer: ccctccagacgcaccggagtcggggg the flow of the air in the air conditioner,
ICP4USr reverse primer:
aagtcgactctagaggatcgatctctgacctgagattggcggcactgaggta
amplifying the upstream flanking sequence of the ICP4 gene;
c. amplification of flanking sequences downstream of the ICP4 gene: using the virus genome DNA obtained in the step a as a template, and using the following ICP4DSf forward primer and ICP4DSr reverse primer:
ICP4DSf forward primer:
aaaagtcgacctgcaggcatgctaacgaggaacgggcagggggc
ICP4DSr reverse primer: aaaaaagcttgcatgcccacgtgcgcggggccagacgggct amplifying the flanking sequence of ICP4 gene;
the upstream and downstream flanking sequences were cloned into the pSP73 plasmid, and pICP4del-eGFP plasmids were constructed: mixing the SalI digested upstream flanking sequence of the amplified ICP4 gene and the SalI/HindIII double digested downstream flanking sequence of the ICP4 gene and connecting the mixture to the EcoRV/HindIII site of pSP73 to obtain pICP4 del; cutting an eGFP expression cassette controlled by a CMV promoter from pcDNA3.1-eGFP by EcoRI/XhoI, filling the tail end by T4DNA polymerase, and inserting the cut end into an EcoRV site of pICP4del to obtain pICP4 del-eGFP;
d. three sequences in the ICP4 gene are amplified by three times of PCR:
first, the following primers were used:
ICP4-1st forward primer: ttttttgaattcatggcgtcggagaacaagcagcgcc
ICP4-1st reverse primer: tggagccaccccatggcctccgcgt
ICP4-2nd forward primer: cgacgccgcgcagcagtacgccctg
ICP4-2nd reverse primer: cggcgggggcgggcccggcgcaccg
ICP4-3rd forward primer: cctcatgtttgacccgcgggccctg
ICP4-3rd reverse primer: ttttttctcgagttacagcaccccgtccccctcgaac
And (b) respectively amplifying three sections of gene fragments ICP4-1st, ICP4-2nd and ICP4-3rd by taking the virus genome DNA obtained in the step a as a template, and then respectively inserting the three sections of gene fragments into the EcoRV site of the pSP73 plasmid to construct the following three plasmids: pSP73-ICP4-1st, pSP73-ICP4-2nd and pSP73-ICP4-3rd, wherein EcoRI and BsrGI are used for cutting out ICP4-1st, BsrGI and PvuI are used for cutting out ICP4-2nd and PvuI and XhoI are used for cutting out ICP-3rd for standby;
e. cutting an hTERTp fragment from a plasmid containing a human telomerase reverse transcriptase promoter hTERTp by using NruI and HindIII, and replacing a CMV promoter cut from pcDNA3-NHN by using NruI and HindIII to obtain a plasmid pcDNA3-NHN-hTERTp, wherein the pcDNA3-NHN is obtained by inserting a NheI-HapI-NheI enzyme cutting site sequence into the NheI site of the pcDNA 3;
f. mixing ICP4-1st, ICP4-2nd and ICP4-3rd obtained in the step d and connecting the mixture to EcoRI and XhoI sites of pcDNA3-NHN-hTERTp obtained in the step e to obtain plasmid pcDNA3-NHN-hTERTp _ ICP 4;
g. digesting the plasmid pICP4del containing the upstream and downstream flanking sequences of the ICP4 gene obtained in the step c by SalI, filling the ends of the plasmid pICP4del, using PmeI and HpaI to cut out an hTERTp _ ICP4 expression cassette fragment from the plasmid pcDNA3-NHN-hTERTp _ ICP4 obtained in the step f, and connecting the fragment with the digested pICP4del plasmid to construct a plasmid pICP4del-hTERTp _ ICP 4;
h. construction of BHK-ICP4 helper cells: cutting the ICP4 gene from the plasmid pcDNA3-NHN-hTERTp _ ICP4 in the step f by using EcoRI and XhoI, and cloning to the EcoRI site and the XhoI site at the downstream of the CMV promoter in the pcDNA3 to obtain a pcDNA3-CMV-ICP4 plasmid; transfecting BHK cells with pcDNA3-CMV-ICP4 plasmids, recombining pcDNA3-CMV-ICP4 plasmid DNA into BHK cell genomes to ensure that some BHK recombined cells obtain resistance to neomycin and express ICP4, killing the non-recombined BHK cells by antibiotic G418, carrying out several rounds of subclone screening, and screening BHK-ICP4 helper cells expressing ICP4 by an RT-PCR method;
(2) knocking out an ICP4 gene promoter in a genome and inserting a granzyme reverse transcriptase promoter hTERTp promoter:
i. culturing herpes simplex virus containing ICP4 gene by using BHK cell, and extracting virus genome DNA;
II, co-transferring the virus genome DNA in the step i and the plasmid pICP4del-eGFP obtained in the small step c in the step (1) into the BHK-ICP4 helper cell obtained in the small step h in the step (1), replacing an ICP4 gene of herpes simplex virus HSV containing an ICP4 gene by a green fluorescent protein GFP expression cassette in the plasmid pICP4del-eGFP through homologous recombination, so that the virus plaques of the recombinant virus emit green fluorescence, carrying out a plurality of cycles of plaque purification, selecting green fluorescent virus plaques, and purifying the recombinant virus HSV-d4 GFP;
culturing HSV-d4GFP virus and extracting genome DNA;
iv, co-transferring the genomic DNA of the recombinant virus HSV-d4GFP and the DNA of the plasmid pICP4del-hTERTp _ ICP4 obtained in the step (1) in the step (g) into a BHK-ICP4 helper cell, and replacing a green fluorescent protein GFP expression cassette of the recombinant virus HSV-d4GFP by the hTERTp _ ICP4 expression cassette through homologous recombination, so that the virus plaques of the new recombinant virus do not emit green fluorescence, selecting non-fluorescent virus plaques through several rounds of plaque purification, and purifying the recombinant herpes simplex virus HSV-hTERTp _ ICP 4;
2) construction of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ Lungca
(1) Synthesizing complementary DNA fragments with the length of 145 bases, namely hp-ICP4 plus and hp-ICP4 minus respectively, according to the following sequences, wherein the N sequence is random base, one side of a flanking known sequence is homologous with a3 'end sequence of a telomerase reverse transcriptase promoter on HSV-hTERTp _ ICP4, and the other side of the flanking known sequence is homologous with a 5' end sequence of an ICP4 gene;
hp-ICP4 positive:
CCGCGAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNGAATTCATGGCGTCGGAGAACAAGCAGCGCCCCGGCTCCCCGGGCCCCACCGACGGGCCG
hp-ICP4 negative:
CGGCCCGTCGGTGGGGCCCGGGGAGCCGGGGCGCTGCTTGTTCTCCGACGCCATGAATTCNNNNNNNNNNNNNNNNNNNNNNNNNGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTCGCGG
(2) constructing a recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA:
extracting HSV-hTERTp _ ICP4 virus genome DNA, carrying out positive and negative homologous recombination on the synthesized DNA fragment hp-ICP4 and hp-ICP4, and screening by using lung cancer cells A549 to obtain HSV-hTERTp _ ICP4_ LungCA with high titer aiming at the lung cancer cells, which is called HSV _ LungCA for short;
(3) culturing and purifying HSV _ LungCA recombinant virus, extracting virus genome DNA, sequencing the combination part of a telomerase reverse transcriptase promoter and an ICP4 gene, and determining that the sequence of 25 bases is AGCAGGACCTCGCCCTTGGGACGAC;
3) construction of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP
(1) Construction of shuttle plasmid pdICP34.5-eGFP
A. Culturing HSV-hTERTp _ ICP4 by using BHK-ICP4 cells, and purifying the genomic DNA of the HSV-hTERTp _ ICP 4;
B. amplifying the flanking sequence of the ICP34.5 gene upstream: and B, taking the virus genome DNA obtained in the step A as a template, and using the following ICP34.5USf forward primer and ICP34.5USr reverse primer:
ICP34.5USf：CTCTGACCTGAGATTGGCGGCACTG
ICP34.5USr：
GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGCTCCTGACCGCGGG
amplifying an upstream flanking sequence of the ICP34.5 gene;
C. amplification of flanking sequences downstream of the ICP34.5 gene: and B, taking the virus genome DNA obtained in the step A as a template, and using the following ICP34.5DSf forward primer and ICP34.5DSr reverse primer:
ICP34.5DSf：
GCGGCCGCAGCGCTGCGGCCGCCAGCGCGGCGGGGCCCGGCCAACCA
ICP34.5DSr：TTCTTCCCTCTTCTCCCGCCCTCCA
amplifying the downstream flanking sequence of the ICP34.5 gene;
D. connecting the upstream and downstream flanking sequences of the ICP34.5 gene: taking the ICP34.5 upstream and downstream flanking sequences obtained in the steps B and C as templates, and amplifying and connecting the ICP34.5 gene upstream and downstream flanking sequences by using ICP34.5USf and ICP34.5DSr;
E. cloning the upstream and downstream flanking sequences onto pSP72 plasmid to construct pdICP34.5;
F. obtaining an eGFP expression sequence from pcDNA3.1-eGFP and inserting the eGFP expression sequence into an AfeI site of pdICP34.5 to obtain pdICP34.5-eGFP;
(2) construction of HSV-hTERTp _ ICP4_ LungCA-GFP
Culturing HSV-hTERTp _ ICP4_ LungCA (HSV _ LungCA) by using A549 cells, and purifying the genomic DNA of the HSV-hTERTp _ ICP4_ LungCA; the genome DNA and the plasmid pdICP34.5-eGFP in the F in the step (1) are co-transferred into a lung cancer A549 cell, and through homologous recombination, a green fluorescent protein GFP expression box in the plasmid pdICP34.5-eGFP replaces an ICP34.5 gene of herpes simplex virus HSV _ LungCA containing the ICP34.5 gene, so that the virus plaques of the recombinant virus emit green fluorescence, and through several rounds of plaque purification, the green fluorescent virus plaques are selected, and the recombinant virus HSV-hTERTp _ ICP4_ LungCA-GFP, which is called HSV _ LungCA-GFP for short, is purified.
2. The use of the recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP prepared by the method of claim 1 in the preparation of a diagnostic kit for lung cancer.
3. The method of claim 2Use, characterized in that; the diagnostic kit consists of recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP, human CD45 antibody marked by PEcy5 and erythrocyte lysate; wherein the content of the herpes simplex virus HSV-hTERTp _ ICP4_ LungcA-GFP is 5E6-5E7CCID50The content of the PEcy 5-labeled human CD45 antibody is 0.1-0.5 mg/ml; the erythrocyte lysate contains ammonium chloride, disodium ethylene diamine tetraacetate, potassium bicarbonate and ultrapure water, wherein the content of the ammonium chloride is 0.10-0.20M, the content of the disodium ethylene diamine tetraacetate is 0.5-2 mM, and the content of the potassium bicarbonate is 5-15 mM.
4. Use according to claim 3, characterized in that: the content of the herpes simplex virus HSV-hTERTp _ ICP4_ LungcA-GFP is 5E6-5E7CCID50The content of the PEcy5 labeled human CD45 antibody is 0.2 mg/ml; in the erythrocyte lysate, the content of ammonium chloride is 0.15M, the content of ethylene diamine tetraacetic acid is 1mM, and the content of potassium bicarbonate is 10 mM.
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