CN111440811A - Cervical cancer vaccine simultaneously targeting HPV 16L 1 and HPV16E5E6E7 and preparation method thereof - Google Patents

Abstract

The invention discloses a cervical cancer vaccine simultaneously targeting HPV 16L 1 and HPV16E5E6E7 and a preparation method thereof, wherein HPV 16L 1 protein coded by SEQ ID NO: 1 and E5, E6 and E7 antigen peptides are combined to prepare the cervical cancer vaccine, bioinformatics analysis is utilized to screen long peptide fragments with higher scores for main human H L A types, and an E5E6E7 polypeptide fragment with the strongest immune effect is obtained by combining in-vitro tests to form a double-effect vaccine core, and an insect cell-baculovirus expression system is applied to prepare the L1-E5E 6E7 cervical cancer vaccine, so that the cervical cancer vaccine has dual effects of prevention and treatment.

Description

Cervical cancer vaccine simultaneously targeting HPV 16L 1 and HPV16E5E6E7 and preparation method thereof

One, the technical field

The invention belongs to the field of tumor immunotherapy.

Second, background Art

The WHO data show that 45 ten thousand new cases of cervical cancer occur every year in the world, and more than 80% of the new cases occur in developing countries, China is the second highest developing country of cervical cancer which is second to chile, although nine-valent, four-valent and two-valent cervical cancer preventive vaccines have been developed internationally, the preventive vaccines only can take effect on people WHO do not have HPV infection yet, the simple preventive treatment of patients WHO continuously infect HPV for a long time cannot solve the practical problem, the research and development of specific therapeutic vaccines are urgent, in addition, most of cervical cancer patients and postoperative recurrent patients are caused by HPV integration infection, the preventive vaccines have no obvious effect on the integrated HPV infection, most of cervical disease patients WHO relapse after the operation are caused by HPV integration infection, and the preventive vaccines of L1 in the market have no capacity to treat the integrated HPV infection and are urgent clinical research.

The HPV infection is a primary carcinogen and an important promoter of cervical cancer, and is well recognized, so far, it has been found that one hundred or more HPV types of different types are classified into high-risk type and low-risk type according to pathogenic characteristics, 99% of cervical cancer is related to high-risk type HPV, wherein nearly 60% of cervical cancer is caused by HPV16 infection, HPV early region (E region) gene encoding proteins such as E1, E2, E4, E5, E6, E7, wherein E1, E2 are related to viral replication and transformation, E4 may be involved in virus release from infected cells and partial viral packaging, E5 protein is a minimal transforming protein which plays a role in promoting malignant transformation of cells and is involved in early stage of primary lesion inhibition of HPV infection in early stage of HPV infection and is significantly reduced by HPV apoptosis in malignant transformation cycle of infected cells, and Rb 2 is a major factor of HPV infection, 3614, HPV infection is considered to be a major malignant transformation cycle, HPV infection, HPV early stage of HPV infection, HPV infection is considered to be a major malignant transformation cycle, and the HPV infection of HPV infected cells is considered to be a major malignant transformation cycle, and the malignant transformation of HPV infected cells such as a high-apoptosis-inducing HPV, and the HPV infection of HPV infected cells, the HPV early stage of HPV, the HPV infection of HPV, the HPV early stage of HPV, the HPV infection of HPV, the HPV infection of HPV, the HPV early stage of HPV, the HPV infection of HPV, the HPV infection of HPV, the HPV early stage of HPV, the HPV infection of HPV, the HPV infection of HPV, the.

The HPV 8 proteins L, E1 and E4 cannot naturally immunize infected persons, and L will have 50% -100% of infected persons who will generate protective antibodies in weeks or years after infection, which is one of the bases of HPV prophylactic vaccine preparation, E5 can mediate immune tolerance by inhibiting MHC-1 expression, E7 is related to delayed hypersensitivity reaction in precancerous lesion stage, however, the vaccine against E6 and E7 can only inhibit HPV-related diseases by 80%, and completely block these two "classical" targets still cannot completely eradicate HPV malignancy, which compels us to carry out deeper thinking, based on HPV pathogenic mechanism and natural immunogenicity, whether there are other key factors besides E6 and E7 in HPV pathogenic process, and whether E5 has the same malignant potential before HPV integration is just a convincing target, whether there are other key factors in addition to E6 and E7, and whether there are other key factors in HPV pathogenic target sites, and E5 has the same potential for HPV integration, thus the HPV is a reliable target for HPV infection, and the host cell-induced by HPV infection, the host cell targeting HPV receptor antigen binding, thus the HPV 3, the HPV epitope, the HPV receptor can be induced by HPV 3, the host cell-induced HPV epitope, the HPV 3-HPV infection is a similar to the HPV infection, the HPV infection can be induced by the HPV infection, the HPV infection can be induced by the host cell-induced by the host cell infection, the HPV infection, the host cell can be induced by the HPV infection, the HPV infection can be induced by the HPV infection, the HPV infection can be induced by the host can be induced by the HPV infection, the HPV infection can be induced by the HPV infection, the HPV infection by the HPV.

Third, the invention

Based on the technical background and the important medical requirements, the invention discloses a screening, verification and preparation method of a cervical cancer vaccine which simultaneously targets three key pathogenic factors (target points for treating cervical cancer) of HPV16E5E6E7 and HPV 16L 1 (target points for preventing cervical cancer) with dual effects of prevention and treatment, and hopes to effectively solve the limitation of the current HPV16 positive tumor treatment through the vaccine with the effects of prevention and treatment and lay the foundation for developing the cervical cancer vaccine of Chinese for preparing the Chinese.

L1 (target point for preventing cervical cancer) is combined with E5E6E7 long peptide, a bioinformatics platform is utilized to analyze and screen peptide segments aiming at the antigen epitope of human major histocompatibility complex MHC molecule (H L A), meanwhile, Bac toBac insect cell-baculovirus expression system is safe and efficient, large-segment foreign genes can be loaded, the advantages of similarity to natural protein in structural function, antigenicity, immunogenicity and the like of an expression product are utilized, and the research content of synthesizing L1 + E5E6E7 has no similar report at home and abroad.

The invention adopts the following technical scheme:

a group of immune antigen peptide coding nucleic acid sequences consists of three nucleic acid fragments which respectively code E5: F LL CFCV LL, E6: K L PQ L CTE L and E7: YM L D L QPET.

The coding nucleic acid sequences of the three peptide fragments of the HPV16E5, E6 and E7 are used for preparing the tumor and the preparation for researching the prevention and treatment of HPV16 related diseases.

The invention further provides a vaccine preparation, which is a cervical cancer vaccine preparation prepared by combining the HPV 16L 1 protein coded by the SEQ ID NO. 1 and the antigen peptides E5, E6 and E7.

The invention further provides a method for preparing the chimeric virus-like particles cV L Ps, which comprises the steps of expressing the three nucleic acid fragments and HPV 16L 1 shown in SEQ ID NO: 1 by using an insect cell-baculovirus expression system, and finally obtaining the HPV 16L 1+ "E5E 6E 7" chimeric virus-like particles cV L Ps through construction, verification, amplification and purification respectively, wherein the packaging amplified HPV 16L 1V L P is used for comparison research.

The method for preparing cV L Ps as described in claim above, wherein the method comprises cloning HPV 16L + E5E6E7 gene by PCR method using Bac to Bac baculovirus expression system, connecting pFastHTb to pFastHTb 16L + "E5E 6E 7" after sequencing identification with linearized pFastHTb, transposing and recombining pFastHTb HPV 16L + "E5E 6E 7" with DH10BAC competent bacteria, screening and recombining Bacmid HPV 16L + "E5E 6E 7" clone by resistance and blue white spots, extracting the virus with infectivity obtained by transfecting insect cell Sf-9 with Bacmid HPV 16L + "E5E 6E 7", infecting Sf-9 cell with virus to express protein expression and amplification, identifying by RT-PCR and Western blot, and screening cV 7 expressing HPV16 type HPV 3 + "E5E 6E 7375" 4642 ".

Wherein the sequences of the upstream primer and the downstream primer of HPV 16L 1 are L1-P1: 5'

CGGAATTCAAATGTGCCTGTATACACGGGTC 3'；L1-P2：5'

CGCGGATCCGCGCACACATATTACTTCCTGG 3'。

The invention also provides the chimeric virus-like particle cV L Ps prepared by the method.

The invention also provides application of the cV L Ps prepared by the preparation method of the chimeric virus-like particles cV L Ps in preparation of a cervical cancer vaccine with both prevention and treatment effects.

The invention also provides application of the cV L Ps prepared by the preparation method of the chimeric virus-like particles cV L Ps in preparation of reagents for cervical cancer and immunotherapy.

The invention achieves the following effects:

1. successfully screening and selecting HPV16E5, E6 and E7 CT L epitope polypeptides of human H L A-A2, and preparing and constructing an HPV 16L 1+ "E5E 6E 7" cervical cancer vaccine preparation by using an insect cell-baculovirus expression system, and injecting a new idea for preventing and treating cervical cancer;

2. discussing host immune microenvironment change in HPV16 infection process, providing new experimental evidence for comprehensively describing pathogenic mechanism of HPV16, and laying a solid foundation for Chinese developing cervical cancer vaccine.

Description of the drawings

FIG. 1 is a screening of HPV16E5 polypeptide fragment against human H L A-A2.

FIG. 2 is a screening of HPV16E 6 polypeptide fragments against human H L A-A2.

FIG. 3 is a screening of HPV16E 7 polypeptide fragments against human H L A-A2.

FIG. 4 PCR amplification of HPV 16L 1-E5E6E7 fusion gene fragment 1: L1-E5E 6E7 fusion gene band visible around 1.5kb, consistent with the predicted size, 2: negative control, M: 1kb ladder DNA marker.

FIG. 5 is a diagram showing that T-HPV 16L 1-E5E6E7 recombinant plasmid is identified by EcoRI and Hind III double digestion, No. 2, 3, 5 recombinant plasmid contains T vector 3.0kb and L1-E5E 6E7 fusion gene fragment 1.5kb, indicating successful ligation, No. 1,4 plasmid does not contain L1-E5E 6E7 fusion gene, indicating no ligation, and M: 1kb ladder DNA marker.

FIG. 6 shows the PCR technology to identify recombinant bacmid-HPV 16L 1-E5E6E7.1 as negative control, 2-3 shows the amplification of two primers of L1-E5E 6E7 fusion gene to obtain target gene segment of about 1.5kb, 4-5 shows plasmid DNA of bacmid-HPV 16L 1-E5E6E7 and M1 shows Marker VI.

FIG. 7 shows that after Bacmid-HPV 16L 1+ "E5E 6E 7" DNA transfects insect cell Sf-9 (inverted microscope), A-C is normal Sf-9 insect cell, D-F is Bacmid-HPV 16L 1+ "E5E 6E 7" recombinant baculovirus infects Sf-9 insect cell, the cell volume is gradually increased, and a plurality of particles appear in the cell and are easy to shed.

FIG. 8 uses PCR technology to identify recombinant bacmid-HPV 16L 1-E5E6E 7: 1: the visible band at about 4kb, indicating successful translocation.2-4: using L1-E5E 6E7 fusion gene two primers to amplify, about 1.5kb, the purpose gene fragment, indicating that the bacmid-HPV 16L 1-E5E6E7 recombinant plasmid contains L1-E5E 6E7, 5: negative control, M: 1 kblader DNA marker.

FIG. 9 SDS-PAGE detection of purified HPV 16L 1-EE5E6E7 fusion protein HPV 16L 1-E5E6E7 fusion protein Sf-9 cells 1, HPV 16L 1-E5E6E7 fusion protein purified under non-denaturing conditions 3 fusion protein treated with binding buffer 4 unpurified fusion protein.

FIG. 10 Western blotting analysis of HPV 16L 1+ "E5E 6E 7" protein expression in Sf-9 cells and 10% SDS-PAGE analysis of HPV 16L 1+ "E5E 6E 7" protein purification

FIG. 11 Electron microscopy of Sf-9 cells transfected with recombinant Bacmid DNA before purification and purified viral particles

FIG. 12 is a graph showing the results of mouse hemagglutination assay.semi-quantitative analysis was performed using Image-Pro + v6.0(Media Cybernetics, Inc) and SPSS 13.0. the mean value of (P0.000) each bar represents the results of three experiments was obtained by successful hemagglutination of pure HPV 16-L1 + E5E6E7cV L P protein at different concentrations (25-400 ng) in vitro.

FIG. 13 construction of recombinant baculovirus with Bac-to-Bac baculovirus expression System and expression of target Gene

FIG. 14 restriction sites and multiple cloning sites of pFastBac-Htb expression vector

FIG. 15 multiple cloning site sequence of pFastBac-Htb expression vector

FIG. 16 PCR identification of recombinant bacmid-HPV 16L 1+ E5E6E7 DNA and translocation schematics

Figure 17 prevention group: non-tumorigenic condition of mice in each group after dry prognosis of different cervical cancer vaccine preparations

Figure 18 prevention group: different cervical cancer vaccine preparations for dry prognosis of tumorigenesis of mice in each group

Figure 19 treatment groups: different cervical cancer vaccine preparations for dry prognosis of tumorigenesis of mice in each group

FIG. 20E L ISA results show that in cV L P group, V L P group has obviously enhanced IFN gamma expression compared with each control group I L-2

Fifth, detailed description of the invention

Example 1 selection of a selected HPV16E5, E6, E7 epitope fragment to human H L A-A2

The full-length sequences of HPV16E5, E6 and E7 are analyzed by a biological information and molecular structure analysis system to obtain corresponding fragments, and the target fragments with higher scores, strong affinity, better immunogenicity and antigenicity are selected as candidate fragments to be subjected to next laboratory screening, the results are shown in a figure 1-3, and the antigen Epitope fragments with the best effect obtained by final screening are E5: F LL CFCV LL, E6: K L PQ L CTE L, E7: YM L D L QPET (Epitope-predition (http:// www.Syfpeithi.de /) (Rammenseee et al, 1999), ProPred-I (http:// www.imtech.res.in/Raghava/ProPred1/index. html) (Singh and Raghava,2003), figures 1-3

Example 2 construction of HPV 16L 1+ "E5E 6E 7" short-acting cervical cancer vaccine formulation Using insect cell-baculovirus expression System

Recombinant baculovirus was constructed and the gene of interest was expressed using the Bac-to-Bac baculovirus expression system according to the procedures described in figure 11. The specific operation steps are as follows:

1) amplification of fragments of HPV 16L 1+ "E5E 6E 7" interest

The HPV16 type complete genome sequence is analyzed by using Oligo 6.0 primer design software, a pair of primers (P) for amplifying HPV 16L 1 full length and an E5.E6.E7 epitope fragment screened by bioinformatics prediction analysis and in vitro and in vivo effect experiments are designed, and the specific sequences are as follows (the primers are synthesized by Shanghai Yingjun limited Biotechnology).

Upstream and downstream primer sequences of HPV 16L 1

For cloning convenience, EcoR I or Hind Ш cleavage sites and protective bases are added to the 5' ends of the two primers, respectively.

E5.E6.E7 epitope fragment

The reaction reagents were as follows:

1. the primer is prepared into 10-50 umol/L with deionized water, and the dosage is 1-2ul/100ul during amplification.

2. 5U/ul Taq polymerase, and the dosage of the Taq polymerase during amplification is 2-5U/100ul.

3, PCR reaction buffer solution is provided with Taq enzyme,

4. dNTPs-neutralization mixture (ZM, 10X) 10mg each of dATP, dGTP, dCTP and dTTP sodium salt was dissolved in 2ml of deionized water, adjusted to pH7.0-7.5 with O.1M Na0H, dispensed, and stored at-20 ℃ at a concentration of 5 mM.

5. A plasmid as a template was used as a gift for the Provisions of the EthelMieheledeVilliers university of Heidelberg, Germany. (the complete sequence of HPV16 in the template is as follows:

we have carried out relevant studies with this plasmid and published articles Shujie L iao, Dongrui Deng, WeinaZhang, Xiaoji Hu, Wei Wang, Hui Wang, Yunning L u, Shixuan Wang, L i Meng, Ding Ma. Human pallomavir 16/18E5 proteins scientific cancer cell promotion, migration and migration in video and access in video growth, 29(1), 95-102,2013 (SCI)

TABLE 1 reaction system (50. mu.l mix on ice)

Gently mixing, centrifuging, placing on a PCR instrument, starting the following reaction,

TABLE 2 amplification conditions

Identification of HPV 16L 1 sequence by 1% agarose gel electrophoresis

(l) The gel preparation comprises loading 1XTAE electrophoresis buffer 20ml and 0.2g agar sugar powder into a Erlenmeyer flask. The agarose was dissolved by heating in a microwave oven and the solution was cooled to 60 ℃. Placing a comb at a distance of 0.5-1.0mm from the bottom plate, pouring warm agarose solution into a rubber mold with a gel thickness of 3-5mm, and checking whether air bubbles exist under or between teeth of the comb. After the gel has completely solidified (30-45 min at room temperature), the comb is carefully removed, the gel is placed in an electrophoresis bath, and a sufficient amount of electrophoresis buffer just about lmm deep below the gel surface is added.

(2) Electrophoresis, after mixing the PCR product with 10 Xloading buffer, the mixture was slowly added to the sample tank using a disposable micropipette, in which 1KB DNA ladder was added to one lane. And covering the electrophoresis tank and electrifying, wherein the anode is red, and the sample electrophoreses towards the anode. Electrophoresis with a voltage of 100 mV. Electrophoresis to remove the sea phenol blue and xylene blue FF in the gel for the appropriate distance. The power was turned off, the gel was removed, and the gel was soaked in ethidium bromide (10 mg/ml stock solution prepared with double distilled water) to a final concentration of 0.5. mu.g/m 1 for 20-30 minutes with the result being observed under an ultraviolet lamp.

(3) The ultraviolet photographic system takes pictures.

As can be seen from the results in FIG. 4, a band of the fusion gene L1-E5E 6E7 was observed at around 1.5kb, which agrees with the predicted size, and no band was observed in the negative control.

2) Identification and sequencing of fragments of HPV 16L 1+ E5E6E7 mesh

① purification of PCR amplification products (HPV 16L 1+ E5E6E7 destination fragment)

The PCR amplification product was purified by using a PCR product purification kit (U-gene) by the following specific procedures:

A. taking 80 mu l +400 mu l PB of PCR amplification products, and mixing uniformly;

B. placing in a centrifugal column, and centrifuging for 30-60 sec;

C. discarding the waste liquid in the waste collection pipe, and putting the centrifugal column into the pipe;

d, +750 μ l PE, centrifugate for 10 min;

E. discarding waste liquid in the waste collecting pipe, and centrifuging for 10 min;

F. the spin column was put into a 1.5ml Eppendorf (Ep) tube;

G.+ddH₂placing O30 μ l into the center of the column, and centrifuging for 1 min;

H. and measuring the OD value.

② enzyme-cutting purified product

TABLE 3

Mixing, 37 deg.C overnight

③ 1% agarose gel electrophoresis followed by gel purification the digestion product was purified using a gel purification kit (U-gene) as follows:

A. cutting a mesh strip under an ultraviolet lamp, putting the cut mesh strip into a 1.5ml EP tube, and weighing;

B. adding 1ml GS buffer, 50 deg.C, 10 min;

C. putting the melted gel into a centrifugal column tube, centrifuging at 10000rpm for 30sec, and removing waste liquid;

d, + 500. mu.l wash buffer, centrifuging at 10000rpm for 30sec, repeating once;

E. centrifuging at the highest speed for 1min to discard the wash buffer;

F. placing the column into a sterile 1.5ml Ep tube;

G.+ddH₂placing O30 μ l in the center of the column, standing at room temperature for 2-10min, and centrifuging at the highest speed for 1 min;

the column was removed and 1.5ml Ep tubes were stored at-20 ℃.

④ tailing reaction (sticky A ends added):

TABLE 4

Mixing at 72 deg.C for 20min

⑤ to be connected to

-T Easy vector

TABLE 5

Mixing, 22 deg.C, 2h, and electrophoresis analysis and identification.

⑥ transformation of DH5 α bacteria and blue-white screening

Preparation of a competent DH5 α bacteria:

1) reagents and materials:

l B liquid culture medium containing no antibiotic, 0. lmol/L CaCl₂The preparation method of the solution is shown in appendix E coil DH5 α single colony or frozen strain (such as JM 109).

2) The process comprises the following steps:

a. recovering strain by inoculating 100 μ l strain into 3m 1L B glass test tube;

b, shaking overnight at the temperature of 200rpm-250rpm x37 ℃ for 12h-16 h;

C. taking 100 mu l of the bacterial liquid the next day to a test tube containing 10ml of L B;

d.37 ℃ shaking vigorously for 2-3 h;

e. taking out when OD600 value reaches 0.3-0.4, and immediately placing in ice bath for 10-15 min;

sterile operation is required from the step;

f. transferring the bacteria into a precooled 10ml plastic centrifuge tube;

g. centrifuging at 4 deg.C for 4,000g x10 min;

h. abandoning the culture solution, inverting the tube on the filter paper and sucking the residual culture solution by using a sterile gun head;

i. adding pre-cooled 0. lmol/L CaCl₂2ml of heavy suspension thallus;

j. placing in ice bath for 30 min;

k. centrifuging at 4 deg.C for 4,000g X10 min;

1. discarding the supernatant, inverting the supernatant on filter paper, and sucking residual culture solution by using a sterile gun head;

m. 400. mu.l of precooled 0. lmol/L CaC1₂Resuspending the cells (light weight), and packaging into 200 ul/tube or 50 μ l/tube;

n, placing in a refrigerator at 4 ℃ for 12-24 hours, and then using for transformation, or adding glycerol and freezing at-70 ℃ for standby.

O. cryopreservation of competent cells, subpackaging competent cells into 200 μ 1/tube; 30% volume (80. mu.1) of glycerol stock solution was added to each portion. Standing at 70 deg.C for 3 months.

B, transformation:

1) reagents and materials:

1. competent DH5 α bacteria, Amp resistant plates (solid medium formulation and plating see appendix);

water bath 42 ℃;

2.I PTG stock solution(0.1M):

2g IPTG(Promega)

50ml double distilled deionized water

Filtering, sterilizing, and storing at 4 deg.C.

X-Gal (100 mg X-Gal (Promega)) was dissolved in 2ml of xylenol amine (N N-dimethyl-formamide), and the mixture was stored under dark at-20 ℃ with sealing with aluminum foil.

4, L B medium:

5. l B plates containing ampicilin/IPTG/X-gel L B plates were prepared as described in the appendix, ampicilin was added and the plates were inverted after addition of 0.5mM IPTG and 80ug/m1X-Gal or 100ul100mM IPTG and 20ul5Omg/ml X-Gal were added to the plate surface and used after complete uptake at 37 ℃ for 30 min.

2) Procedure (according to pGEM-Teasy instructions):

a) rapidly taking out DH5 α bacterial susceptibilities from a refrigerator at-80 ℃ (50 ul split charging);

b) add 5. mu.l of the above ligation product T-HPV 18L 1+ E5E6E 7;

c) mixing, standing on ice for 30 min;

d) heat shock in 42 ℃ water bath for 90 sec;

e) adding 200 μ l L B, shaking at 37 deg.C and 150rpm for 1 h;

f) take 80. mu.l of the plate (L B plate containing ampicillin/IPTG/X-gel);

g) at 37 ℃ overnight;

h) after observing the growth of blue-white spots, the plates were stored at 4 ℃ (blue-white spots are more clear).

C, picking a plurality of independent round white bacterial plaques for small-scale culture.

The operation procedure is as follows:

1. a sterilized 5ml culture tube was added with 3m 1L B medium.

2. 3-5ul of 5Omg/ml ampicillin (if host bacteria are cultured, antibiotics are not added), or the antibiotics are added into L B culture medium.

3. Taking single colony with aseptic toothpick (or gun head), feeding into culture solution (or taking 100ul of bacteria solution, adding into culture solution), and sealing the tube with aseptic cotton ball.

Shaking culture at 4.37 deg.C for 100-.

5. Preservation of the strain, which is to take 1.5ml of centrifuge tube and add 0.3ml of preservation solution (100% glycerol). 0.7ml of the bacterial solution was added, mixed well and stored at-70 ℃.

Small extraction of D plasmid (application of Boda Take plasmid small extraction kit)

1.5ml of the bacterial solution was placed in an Ep tube and centrifuged at 10000rpm for 30 seconds. Discard the supernatant and control it completely.

Add solution I100U L, resuspend the cells, and shake vigorously.

150ul of the solution II is added, and the mixture is mixed by turning upside down for 5 times.

Add 150ul of solution III and mix well.

The mixture was left at room temperature for 5min at 12000rpm and centrifuged for 10 minutes.

Add 420ul binding buffer to the centrifugation and adsorption column, transfer the supernatant to the centrifugation and adsorption column, mix well, 12000rpm, centrifuge for 30 sec. And (4) pouring the waste liquid in the collecting pipe.

750ul of rinsing buffer was added to the centrifugal adsorption column, and the column was left to stand at 12000rpm for 30 sec. And (4) pouring the waste liquid in the collecting pipe.

This was repeated once, and after the waste liquid in the collection tube was discarded, it was centrifuged at 12000rpm for 2 minutes.

The centrifugal adsorption column was taken out, put into another 1.5ml Ep tube, 50ul of sterile deionized water was added thereto, and after standing at room temperature for 5 minutes, it was centrifuged at 12000rpm for 2 minutes.

The plasmids were collected. The plasmids were stored at 4 ℃ or-20 ℃. TAE was used as a buffer, and the mixture was electrophoresed at 15OV and 60mA for 30 minutes to observe the presence of the plasmid.

⑦ double restriction enzyme (EcoRI and Hind III) to identify T-HPV 16L 1+ E5E6E7

TABLE 6

Mixing, at 37 deg.C for 2-6h, and performing electrophoresis analysis.

⑧ sequencing and identification of HPV 16L l + E5E6E7, namely observing an electrophoresis result, selecting clone connected with a target band, carrying out small-amount amplification, and sending a suspension to Invitrogen company for sequencing.

3) Construction of recombinant shuttle plasmid pFastBacHTb-HPV 16L 1+ E5E6E7

The specific structure of the plasmid is shown in FIG. 13, and the multiple cloning site of the plasmid is shown in FIG. 14. The specific operation steps are as follows:

① A small amount of T-easy-HPV 16L 1+ E5E6E7 plasmid and pFastBac-Htb plasmid were extracted and subjected to double-enzyme cleavage.

The restriction enzyme system is that 50ul volume contains 1-2ug DNA, T-HPV 16L 1+ E5E6E7 plasmid and pFastBac-Htb shuttle vector are double-digested by EcoRI and Hind III.

TABLE 7

Slightly centrifuged and mixed. Water bath or incubator at 37 deg.c, mixing overnight, and electrophoresis analysis.

② purification and recovery of glue (U-gene kit, see 2) ③)

③ determination of the concentration of the recovered product, 5. mu.l of the DNA solution was pipetted and dissolved in 200. mu.l of ddH₂O, the 0D value was measured by ultraviolet spectrophotometry, and OD260(DNA concentration) was read, where the DNA concentration (μ g/. mu.l) ═ OD260X5

④ ligation of the recovered HPVI 6L 1+ E5E6E7 sequence and the pFastHTb sequence

The connecting system is shown in Table 8

Mixing, RT, 3h, electrophoresis analysis and identification.

⑤ transformation of DH5 α bacteria, selection of anti-Gen and anti-Amp clones.

⑥ restriction enzyme identification, after bacteria shaking and small-quality-improved plasmid DNA (a plasmid small-quantity extraction kit of Boda Tak, the operation is strictly carried out according to the instruction), EcoR I and Hind III double restriction enzyme identification, the objective fragment of 1.5KB can be obtained by restriction enzyme according to the electrophoresis result, the successful connection is proved, the objective fragment is named as pFastHTb-HPV 16L l E5E6E7, and the positively cloned strain is preserved, the results of figure 5 show that the recombinant plasmids No. 2, 3 and 5 contain a T vector 3.0KB and a L1-E5E 6E7 fusion gene fragment 1.5KB, which indicate that the connection is successful, and the plasmid No. 1 and 4 does not contain a L1-E5E 6E7 fusion gene, which indicates that the connection is not connected.

4) Transposition, construction and identification of recombinant baculovirus bacmid-HPV 16L 1+ E5E6E7

After the pFastHTb-HPV 16L 1+ E5E6E7 is correctly connected through enzyme digestion identification, the pFastHTb-HPV 16L + E5E6E7 is transferred into competent DH10Bac escherichia coli to be subjected to homologous recombination with baculovirus DNA (bacmid) to form bacmid-HPV 16L 1+ E5E6E7, and the identification and the screening are carried out through a PCR technology.

① the plasmid pFastHTb-HPV 16L 1+ E5E6E7 was extracted.

② preparation of DH10Bac competent cells:

a. recovering strain by inoculating 100ul strain into glass test tube containing 3ml SOC culture medium;

b, shaking at 37 ℃ for 12-16 h at 200rpm-250rpm for overnight;

C. taking 100ul of bacterial liquid to a test tube containing 10ml of SOC the next day;

d. shaking vigorously for 2-3 h;

e. taking out when OD600 value reaches 0.3-0.4, and immediately placing in ice bath for 10-15 min;

sterile operation is required from the step;

f. transferring the bacteria into a precooled 10ml plastic centrifuge tube;

g. centrifuging at 4 deg.C for 10min at X4,000 g X10;

h. abandoning the culture solution, inverting the tube on the filter paper and sucking the residual culture solution by using a sterile gun head;

i. adding pre-cooled 0. lmol/L CaCl₂2ml of heavy suspension bacteria;

j. placing in ice bath for 30 min;

k. centrifuging at 4 deg.C for 10min at X4,000 g X10;

l, abandoning the supernatant, inverting the supernatant on filter paper, and absorbing residual culture solution by using a sterile gun head;

m. adding 400ul precooled 0. lmol/L CaCI₂Resuspending the thallus (the operation is light), and subpackaging into 200 ul/tube or 50 ul/tube;

n, placing in a refrigerator at 4 ℃ for 12-24 hours, and then using for transformation, or adding glycerol and freezing at-70 ℃ for standby.

o. cryopreservation of competent cells, subpackaging competent cells into 200 ul/tube; 30% volume (80ul) of glycerol stock solution was added to each serving. Standing at-70 deg.C for 3 months.

The preparation method of SOC culture medium is shown in appendix

③ preparation of plates by preparing plates containing kanamycins (50. mu.g/ml) + gentamycin (7. mu.g/ml) + tetracyclines (10. mu.g/ml) + IPTG (40. mu.g/ml) + X-gel (100. mu.g/ml);

④ transformation

A. Quickly taking out 50 mu l of DH10Bac bacteria competent state from a refrigerator at the temperature of-80 ℃;

B. adding 5 μ l of the above ligation product pFB-HPV 16L 1+ E5E6E7-E7 (50-72aa/4-12 aa);

C. mixing, standing on ice for 30 min;

d.42 ℃ water bath heat shock for 90sec, and then standing for 2min on ice;

E. adding 200 μ l L B, shaking at 37 deg.C and 150rpm for 4 h;

F. taking 80 mu l of planking;

G.37℃，24～48h；

H. after observing the growth of the blue white spots, the culture plate is preserved at 4 ℃ (the blue white spots are clearer);

I. selecting white clone, shaking bacteria, extracting plasmid, converting and plating, and performing blue-white spot selection again.

Under the assistance of DN10Bac host bacteria helper plasmid, pFastHTb-HPV 16L I can transpose with Bacmid of host bacteria, the expression cassette of HPV 16L 1+ E5E6E7 gene and gentamicin resistance gene in the plasmid transposes and contains Bacmid of baculovirus genome through transposition action to recombine, kanamycin (kan), tetracycline (tet) and gentamicin (Gm) antibiotics are selected on L B plate, IPTG and X-gal blue white spot are selected, several independent round white plaques are selected for small-scale culture (recombinant baculovirus is named Bacmid-HPVI 6L 1+ E5E6E7)

⑤ identification

White clone is picked again, plasmid DNA is extracted after shaking, Bacmid-HPVI 8L L DNA is larger than 135 kb, so restriction enzyme digestion identification cannot be carried out, PCR is used for identifying that the target fragment is translocated to Bacmid, two primers PUC/M13 (shown in figure 14) are designed at attTn7 on both sides of lacZ α region of Bacmid, the primer sequences are M13(Forward):5 'GTT TTC CCA GTC ACG AC 3', M13(Reserve):5 'CAG GAA ACA GCT ATG AC 3', synthesized by invitrogen company, if translocation is successful, the size of the PCR product is required to be the size of the primer (2300bp) + the size of the insert fragment, namely about 4000bp, figure 15 shows a principle diagram of PCR identification of recombinant bac-HPV 16L 1+ E5E6E7 DNA and translocation.

TABLE 9 reaction system (50. mu.l)

TABLE 10 amplification conditions

And (3) carrying out 1.0% agarose gel electrophoresis identification.

As can be seen from the results in FIG. 6, 2-3 amplification with two primers for the L1-E5E 6E7 fusion gene succeeded in targeting gene fragments of about 1.5 kb.

5) Obtaining the infectious recombinant baculovirus Bacmid-HPV 16L 1+ E5E6E7

(1) Large scale preparation of Bacmid-HPV 16L 1+ E5E6E7 plasmid for transfection

1) Culturing a large amount of DH10Bac containing Baemid-HPV 16L 1+ E5E6E7 plasmid:

the operation procedure is as follows:

1. l B liquid medium 100-200ml was added to the culture flask and autoclaved.

2. Each ml of L B was supplemented with kanamycin (50. mu.g/ml) + gentamycin (7. mu.g/ml) + tetracyline (10. mu.g/ml)

3. The culture medium is added with 5-10 μ l of the culture solution per ml (final concentration is 0.5-1%).

Shaking culture at 37 deg.C at 200r/min 100-.

2) Mass extraction of plasmids (Steps strict refer to Boda Taike Dati kit instructions)

(2) Sf-9 insect cells were cultured by feeding gentamicin sulfate (500mg/ml) and 10% amphotericin (25. mu.g/ml) at a volume fraction of 10% (V/V) with Grace' S medium containing 10% fetal bovine serum at a culture temperature of 27 ℃.

(3) Cell transfection, namely preparing single cell suspension from Sf-9 cells in logarithmic growth phase and in good state, and mixing 9 × 10⁵Uniformly planting cells in each hole on a six-hole culture plate, culturing at 27 ℃ for more than 1h to ensure complete adherence, wherein the density reaches 5x10⁶The transfection is started. Cell transfection was performed exactly as described by Invitrogen corporation

The specific operation is as follows:

① cells were blown with Grace medium (without antibiotics and FBS) and approximately 9 × 10⁵And placing the cells in a six-hole plate, wherein the volume of Grace culture medium is about 2ml, the used cells are cultured for 3-4 days and are in logarithmic phase, and the survival rate is more than 97%.

② allow cells to adhere for at least 1 h.

③ the following solutions were prepared in 1.5ml sterilized Ep tubes:

a) solution A is 1-2 mu g of recombinant bacmid-HPV 16L 1+ E5E6E7 DNA +100 mu l of serum-free Grace culture medium, and the mixture is mixed evenly.

b) Solution B: 6 μ l

+ 100. mu.l of serum-free Grace medium and mix well.

④ mixing the solution A and the solution B, and incubating for 15-45 min at room temperature.

⑤ the Grace medium in the six-well plate was aspirated, and 200. mu.l of the mixture of solution A and solution B was added, followed by 800. mu.l of serum-free Grace medium, and mixed well.

⑥ 27 ℃ for 5 h.

⑦ the transfection mixture was removed and insect cell culture medium was added, taking close notice of the morphological changes of the cells as follows:

TABLE 11

⑧ transfection for 72h, collecting virus, centrifuging at 3000r/min for 5min, collecting supernatant, getting virus liquid (P1), infecting Sf-9 cells with the first generation virus seed at a ratio of 1:3, harvesting cell supernatant after 72h, getting 2 nd generation infectious Bacnlid-HPV 16L 1+ E5E6E7 virus liquid (P2), which can infect cells directly for expression or preservation.

6) Extraction and PCR identification of bacmid-HPV 16L 1+ E5E6E7 DNA in Sf-9 transfected insect cells

Sf-9 cells were reinfected with virus solution P1 and incubated at 27 ℃ for 3 days. After the mixture of cells and culture medium was centrifuged at 3000rpm for 5min, the supernatant was collected to obtain virus solution (P2), which was stored at 4 ℃ in the dark. The precipitated cells were collected at the same time, FIG. 7.

① extraction of bacmid-HPV 16L 1+ E5E6E7 DNA (genomic DNA miniprep kit)

A. Washing the precipitated cells with PBS buffer solution, centrifuging at 800rpm for 5min, repeating for 1 time, transferring into a 1.5ml Ep tube, centrifuging at high speed for 10sec, adding 100 μ l, oscillating, adding 100 μ l lysis solution and 20 μ l RNaseA/proteinase K solution, rapidly and fully oscillating, mixing, placing in 55 deg.C warm bath for 15min, and reversing the Ep tube back and forth for several times;

B. adding 10 μ l 3M NaAc (pH 4.8), adding 1250 μ l binding buffer solution, shaking thoroughly, mixing, centrifuging, adding the supernatant into centrifugal adsorption column, standing for 1min, and centrifuging at 12000rpm for 30 sec;

C. adding 600 mul of washing cleaning liquid after the waste liquid in the collecting pipe is removed, and centrifuging at 12000rpm for 30 sec;

D. and C1-2 times. The final wash should be centrifuged at 12000rpm for 3min to remove the wash buffer sufficiently;

E. the centrifugation column was carefully removed and put into a clean 1.5ml Ep tube and 50. mu.l of elution buffer was added. After standing for 1min, the mixture was centrifuged at 12000rpm for 3 min. The Ep tube was removed, which was the extracted genomic DNA.

② PCR identification

The HPV 16L 1+ E5E6E7 DNA segment carried in the recombinant baculovirus is amplified by P1 and P2 primers of the HPV 16L 1+ E5E6E7 gene respectively.

Table 12 reaction system: (Total volume 100. mu.l)

Table 13 amplification conditions:

5 μ l of the plasmid was subjected to 1% agarose gel electrophoresis, from the results in FIG. 8, it was found that 1: the desired band was visible around 4kb, indicating successful translocation 2-4: the two primers used for fusion of the gene L1-E5E 6E7 were used for amplification, and that the desired gene fragment was around 1.5kb, indicating that the recombinant plasmid of bacmid-HPV 16L 1-E5E6E7 contained L1-E5E 6E 7.

7) Recombinant virus culture conditions:

sf-9 cells with good growth state are arranged according to the 2x 10⁶Adding each cell/ml into a six-hole plate, wherein the fusion rate of the cells in each hole reaches 70%, pouring the culture solution after 6 hours of adherence, and the ratio of the recombinant virus solution is 10^-3-10^-7After dilution, 500ul of cells in the six-well infected plate are cultured for lh at 27 ℃, the culture solution containing the virus is discarded, and 2ml of 1% low-melting-point agarose is paved in each well. After culturing at 27 ℃ for 7 days, spreading the upper agarose layer containing 0.33% phenol red, and calculating the virus titer after 12 h.

Inoculum 7x10 in 100ml culture flask⁶And (3) adding virus liquid into each Sf-9 cell according to the MOI (mean of the viral infection complex number) of 0.05, 0.1, 0.2 or 0.4, harvesting supernatant at 24, 48, 72, 84, 96 or 120h, centrifuging to remove cell residues, and detecting the virus titer of the supernatant. When the virus is amplified, the cells are infected according to the MOI of 0.2, virus liquid is harvested for 72h, centrifuged, deposited and subpackaged at 4 ℃ for later use.

Inoculum 7X10 in 100ml culture flask⁶(ii) Sf-9 cells, infecting Sf-9 cells with the virus at an MOI of 3, 5, 10, 15, 20 or 30, harvesting the cells at 24, 48, 72, 96 or 120h, respectively, and performing SDS-PAGE, Coomassie Brilliant blueAnd G-250 staining, and detecting the expression condition of the target protein by using a thin-layer scanner.

The amount of virus fluid (ml) required for infection is "predicted MOI value (Pfu/cell) x total number of cells" ÷ virus droplet size (Pfu/ml).

Our experiments showed that at an MOI of 20, the infection efficiency was the highest and the apoptosis rate of the cells was not statistically different from the other groups.

The expression of the fusion protein was successful as shown by SDS-PAGE detection of purified HPV 16L 1-EE5E6E7 fusion protein in FIG. 9.

8) SDS-PAGE detection and Coomassie Brilliant blue G-250 staining

(1) Protein extraction of cultured cells:

A. taking the cells infected with the virus Bacmid-HPV 16L 1+ E5E6E 76 d in the culture plate, and then, 5x10⁵Cells were washed gently 2 times with pre-cooled PBS in an ice bath.

B. Add cell lysate and mix on ice for 20min, and scrape cells carefully on ice with a cell scraper. Transfer to 1ml Ep tube.

C. Low temperature centrifugation (4 ℃)12000rcf x2Omin, supernatant was aspirated and transferred to another lmlEp tube.

(2) Coomassie blue staining and UV spectrophotometry to measure protein concentration.

A. 3ul protein samples were added to 57ul ddH²O, in a test tube, mix well, add 60ul ddH²O is a blank tube.

B. 3ml Coomassie brilliant blue solution is added into each tube, mixed evenly and placed at room temperature for 1 smin. And measuring the absorbance of each tube by using an ultraviolet spectrophotometer, and calculating the protein concentration according to the standard curve.

C. Polyacrylamide gel protein electrophoresis (SDS-PAGE gel):

a. 10ml of separation gel is prepared by the following method:

b. mounting glass plate, rapidly pouring separation gel into the gap, leaving a gel-forming space (tooth length plus 1cm), carefully covering a layer of water on the separation gel solution with Pasteur pipette, and vertically standing the gel at room temperature for 30 min. The cover water was decanted, the gel was washed several times with deionized water to remove the unpolymerized running gum, and the remaining liquid was aspirated with a paper towel.

c. Preparing 4ml of lamination adhesive by the following method:

adding lamination glue, and mounting a comb. The gel was placed vertically at room temperature and after 30min the comb was carefully removed.

d. The desired protein sample was mixed with an equal volume of 2 × SDS loading buffer, placed in an Ep tube and boiled for 10min to denature the protein.

e. The samples were loaded sequentially with a microsyringe, 30ug of protein per sample, added to the bottom and washed in the bottom buffer each time one sample was loaded. Protein molecular weight standards (Marker) were added to the latter well of the sample.

Add l-sense SDS loading buffer into the wells without loading to prevent protein migration.

f. And (4) installing an electrophoresis device, namely connecting the anode with the lower tank, and starting electrophoresis at a voltage of 130 mV. After about 1 hour, the australian phenol blue was observed to reach the bottom of the separation gel and the power was turned off.

D. Coomassie brilliant blue staining was performed for 30min and destained.

a. After electrophoresis, soaking the gel in a dye solution with the volume at least 5 times that of the gel, and slowly rotating the gel for 3 to 4 hours at room temperature in a shaking table.

b. Replacing and recovering dye solution, soaking gel with methanol/acetic acid solution, slowly shaking for 4-5 hr for decolorizing, and replacing the solution for 3-4 times.

c. Decolorization was continued for 24h until satisfactory.

d. The decolorized gel is photographed or dried, or stored in water containing 20% glycerol in a plastic bag for an indefinite period, as shown in FIG. 10.

9) Purification of Bacmid-HPV 16L 1+ E5E6E7 protein, FIG. 10.

Using Ni-NTA His

The Resins purification system purifies HPV 16L 1+ E5E6E7 protein, and the purification steps are as follows:

① purification of main reagents

1x Ni-NTA binding buffer (50 mM/L NaH)₂PO₄pH8.0, 300 mM/L NaCl, 10 mM/L imidazole)

1x Ni-NTA rinse buffer (50 mM/L NaH)₂PO₄pH8.0, 300 mM/L NaCl, 20 mM/L imidazole)

1x Ni-NTA elution buffer (50 mM/L NaH)₂PO₄pH8.0, 300 mM/L NaCl, 250 mM/L imidazole)

② concrete steps

A: taking 0.5ml of 50% Ni-NTA His

Rins +2ml of 1 xNi-NTA combined buffer solution, gently and uniformly mixing, rotating at 300rpm for 3min, removing supernatant, and repeating once;

b: 1.8ml of 1 xNi-NTA binding buffer solution and 200 mul of prepared cell protein are taken, mixed evenly by gentle shaking, bound for 60min at 4 ℃ and multiplied by 1min at 300 rpm;

c: taking the supernatant to a 1.5ml Ep tube for storage;

d: +3ml 1 × Ni-NTA rinsing buffer solution rinsing once, 300rpm × 1min, collecting supernatant and retaining, repeating once;

e: +0.5ml of 1 xNi-NTA elution buffer solution, uniformly mixing by oscillation, carrying out x 1min at 300rpm, collecting and retaining the supernatant, repeating for three times, and sequentially retaining the supernatant;

f, taking 15 ul +2 × SDS-PAGE loading buffer of the supernatant reserved for each time, boiling for 3min at 100 ℃, performing electrophoresis by 10% SDS-PAG, and staining by Coomassie brilliant blue.

10) Observing HPV 16L 1+ E5E6E7V L P before and after purification respectively by a transmission electron microscope and a scanning electron microscope

After sf-9 cells infected with second-generation recombinant viruses 4d are collected and fixed by glutaraldehyde for 3h, the lh is subjected to immersion washing by a barbiturate buffer solution (liquid exchange is carried out for 3 times in the period), then is fixed again by the barbiturate fixing buffer solution, and then is subjected to immersion washing by the barbiturate buffer solution (liquid exchange is carried out for 3 times in the period), then is dehydrated by gradient alcohol (70%, 90% and 100%), is embedded by resin, is polymerized at high temperature, is sliced by an ultrathin slicer, is dyed by uranium acetate and lead xanthate, and virus-like particles in the cells are observed by a transmission electron microscope at different magnification times.

The purified protein under 10 mu L non-denaturing condition is dropped on a copper net coated with a carbon film, and is observed by a scanning electron microscope after phosphotungstic acid counterstaining, the description and the attached figure 11 show that the HPV 16L 1+ E5E6E7V L P can be self-packaged into virus-like particles in vitro (the insertion of the E5E6E7 fragment does not influence the packaging efficiency), and the particles are positioned in the cell nucleus.

11) Verification of in vitro related immune effects of HPV 16-L1 + E5E6E7V L P

(1) Mouse erythrocyte agglutination assay

① collecting blood of C57B L/6 mouse eyeball, centrifuging at 4 deg.C for 1000g x 5min, and collecting erythrocyte;

② the erythrocytes were washed twice with PBS (1000g x 5min) and then resuspended in 1% (v/v) PBS + BSA (1 mg/ml);

③ purified V L P was diluted in PBS + BSA (1mg/ml) fold and mixed with an equal volume of the erythrocyte suspension;

④ mu.l of the mixture (50. mu.l each of V L P and red blood cells) was added to a 96-well plate, two wells for each species, and two-well negative controls (100. mu.l of red blood cell suspension alone without V L P) were set;

⑤ 4 ℃, and the results are observed after 2-3 hours of standing.

⑥ results show that HPV 16-L1 + E5E6E7V L P can cause erythrocytes to agglutinate obviously in vitro, and has strong immunogenicity which is obviously different from that of a control group.

12) Verification of in vivo relevant immune effects of HPV 16-L1 + E5E6E7V L P

Vaccination of human immune reconstituted mice (NCG mice with human immune system reconstitution obtained using human peripheral blood cells (hPBMC)):

selecting 4-6 week old human source immune reconstructed mice, and dividing into two groups for prevention and treatment according to experimental purposes, wherein the former group is divided into HPV 16-L1 + E5E6E7V L P group (cV L P group), HPV 16-L1 group (V L P group), tripeptide group, dipeptide group, single peptide (three groups of E5, E6 and E7), misstructured peptide group (IR-P), and physiological saline group

1) The prevention group comprises administering corresponding preparation to left thigh root of mouse, randomly dividing mouse into nine groups of cV L P group, V L P group, tripeptide group, dipeptide group, E5 group, E6 group, E7 group, hamiltonide group and physiological saline group according to different compatibility, and preparing single cell suspension (1 × 10) from Siha cell in logarithmic phase after one week ⁶100. mu.l) of the mouse, 200. mu.l of the left thigh was inoculated to each mouse, and the corresponding preparation was administered once again, and thereafter, tumor mass formation was observed every 1 to 2 days, each of the constitutional tumor cases is shown in FIGS. 17 and 18, in the cV L P group, the V L P group had a superior tumor preventive effect and almost no tumor formation as compared with the control group.

2) Treatment groups, single cell suspensions (1 × 10) were prepared from each of the logarithmic growth phase Siha cells ⁶100 mul) of the tumor cells were inoculated to the root of the thigh on the left side of each mouse, the corresponding preparation was administered to the root of the thigh on the left side of each mouse one week later, the mice were randomly divided into eight groups, and the mice were randomly divided into nine groups of cV L P group, V L P group, tripeptide group, dipeptide group, E5 group, E6 group, E7 group, misstructured peptide group and physiological saline group according to different combinations, and thereafter, the corresponding preparation was administered once a week every 1 to 2 days to observe the generation of tumor masses, and after the growth of tumor bodies, the tumor sizes were observed and measured 2 to 3 times a week (perpendicularly measuring the maximum diameters a and b of the tumors) until the formation of tumors or 3 to 4 weeks after the treatment, and the tumor volumes were calculated by the formula 1/2 a b.

13)ELISA

A. Collection and preservation of specimens

Blood sampling of mice after eyeballs: the samples were either left at room temperature for 2 hours or at 4 ℃ overnight and centrifuged at 1000x g for 20 minutes, and the supernatant was removed for testing, or the samples were stored at-20 ℃ but repeated freezing and thawing were avoided.

B. Preparation and preservation of reagents

A) Dilution and use of standards.

The kit provides 2 tubes of standard, 10ng per tube, 1 tube per use.

1. Preparing a 10,000pg/ml standard: 1ml of the sample diluent was added to the standard tube, covered and allowed to stand for over 10 minutes, then turned upside down/rubbed repeatedly to aid dissolution.

2. Preparing 1000pg/ml standard: 0.1ml of 10,000pg/ml of the standard was added to an Eppendorf tube with 0.9ml of the sample dilution, mixed and labeled.

3. Preparing 500pg/ml → 15.6pg/ml standard: 6 Eppendorf tubes were prepared, to each of which 0.3ml of sample dilution was added, labeled 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6pg/ml, respectively. 0.3ml of 1000pg/ml standard substance is added into a tube marked with 500pg/ml, and 0.3ml of standard substance is taken out in the same way after being mixed evenly and added into the next tube. And the rest is analogized until the last sample tube.

The dissolved standard should be used within 12 hours.

B) Biotin-labeled antibody working solution.

1. The total amount is calculated according to the required 0.1ml of each hole (0.1-0.2 ml of the total amount is required to be prepared in actual preparation).

2. Working solution is prepared according to the proportion of adding the antibody diluent 99 mu iota into the 1 mu iota biotin-labeled anti-human VEGF. And (4) lightly and uniformly mixing.

C) Preparation of avidin-peroxidase complex (ABC) working solution: prepared within 1 hour prior to use.

1. The total amount is calculated according to the required 0.1ml of each hole (0.1-0.2 ml should be prepared in the practical case).

2. Working solution was prepared at a ratio of 1 μ ι η avidin-peroxidase complex (ABC) to 99 μ ι η ABC diluent. Mix gently.

C. Operating procedure

All reagents were equilibrated at room temperature before use. When the reagent or the sample is diluted, the reagent or the sample cannot be cut and uniformly mixed. A standard curve should be made for each measurement. If the VEGF concentration of the sample is estimated to be > 2000pg/ml, the sample is diluted with a sample diluent in a test tube so that the sample fits within the detection range of the kit.

1. Determining the number of the enzyme label plate holes coated with the antibody required by the detection, adding 1 hole as a TMB blank developing hole, taking the total number as the number of the samples plus 9, performing double detection × 2, and repackaging the rest of the samples and putting the samples in a refrigerator.

2. The standard samples of 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml and 15.6pg/ml are added to a row of 7 wells in sequence, 0.1ml each, and 1 well is added with the sample diluent as a zero well. For human serum, body fluids, tissue homogenates, or cell culture supernatants, 50 μ iota sample dilutions were added to each well followed by 50 μ iota samples.

3. The plate was covered with a lid and reacted at 37 ℃ for 120 minutes.

4. After the reaction, the liquid in the enzyme label plate is absorbed by an automatic plate washing machine (the washing times of the automatic plate washing machine is set to be zero, and then the operation is started); or throwing off the liquid in the enzyme label plate and then beating the enzyme label plate to the absorbent paper for several times. The product is not washed.

5. The prepared biotin anti-human VEGF antibody working solution was added in a volume of 0.1ml per well (except for TMB blank color-developing wells). The reaction was carried out at 37 ℃ for 60 minutes.

6.0.01M TBS or 0.01M PBS 3 times, each soaking for about 1 minutes. Excess liquid is sucked off or thrown off.

7. The prepared ABC working solution was added in a volume of 0.1ml per well (except for TMB blank color wells). The reaction was carried out at 37 ℃ for 30 minutes.

8.0.01M TBS or 0.01M PBS washing 5 times, each soaking 1-2 minutes. Excess liquid is sucked off or thrown off.

9. TMB color development liquid is added into each hole according to 0.1ml, and the reaction is carried out for 20 to 25 minutes in a dark place at 37 ℃ (the front 3 to 4 holes of the standard product can be seen by the flesh eyes at the moment, the gradient blue color is obvious, and the difference between the back 3 to 4 holes is not obvious).

10. TMB stop solution was added in an amount of 0.1ml per well, and the blue color immediately turned yellow.

11. O.d. values were measured at 450nm with a microplate reader, and TMB blank color wells were set as controls.

12. After subtracting the light absorption value of the zero hole from the light absorption values of all the standards and samples, a curve is drawn on a piece of coordinate paper, and the light absorption value is taken as a vertical coordinate and the concentration is taken as a horizontal coordinate.

13. The user may also use various application software to calculate (download) it should be remembered that the actual concentration should be × 2 due to the dilution of the sample by a factor of 1.

As shown in FIG. 20E L ISA, it is confirmed that in cV L P group, V L P group has significantly enhanced expression of TH-1 cytokines such as IFN gamma compared with each control group I L-2, and can cause good CT L effect and kill tumor cells.

Sequence listing

<110> Wuhan Kaideweis Biotech Co., Ltd

<120> cervical cancer vaccine simultaneously targeting HPV 16L 1 and HPV16E5E6E7 and preparation method thereof

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>1518

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atgtctcttt ggctgcctag tgaggccact gtctacttgc ctcctgtccc agtatctaag 60

gttgtaagca cggatgaata tgttgcacgc acaaacatat attatcatgc aggaacatcc 120

agactacttg cagttggaca tccctatttt cctattaaaa aacctaacaa taacaaaata 180

ttagttccta aagtatcagg attacaatac agggtattta gaatacattt acctgacccc 240

aataagtttg gttttcctga cacctcattt tataatccag atacacagcg gctggtttgg 300

gcctgtgtag gtgttgaggt aggtcgtggt cagccattag gtgtgggcat tagtggccat 360

cctttattaa ataaattgga tgacacagaa aatgctagtg cttatgcagc aaatgcaggt 420

gtggataata gagaatgtat atctatggat tacaaacaaa cacaattgtg tttaattggt 480

tgcaaaccac ctatagggga acactggggc aaaggatccc catgtaccaa tgttgcagta 540

aatccaggtg attgtccacc attagagtta ataaacacag ttattcagga tggtgatatg 600

gttgatactg gctttggtgc tatggacttt actacattac aggctaacaa aagtgaagtt 660

ccactggata tttgtacatc tatttgcaaa tatccagatt atattaaaat ggtgtcagaa 720

ccatatggcg acagcttatt tttttattta cgaagggaac aaatgtttgt tagacattta 780

tttaataggg ctggtactgt tggtgaaaat gtaccagacg atttatacat taaaggctct 840

gggtctactg caaatttagc cagttcaaat tattttccta cacctagtgg ttctatggtt 900

acctctgatg cccaaatatt caataaacct tattggttac aacgagcaca gggccacaat 960

aatggcattt gttggggtaa ccaactattt gttactgttg ttgatactac acgcagtaca 1020

aatatgtcat tatgtgctgc catatctact tcagaaacta catataaaaa tactaacttt 1080

aaggagtacc tacgacatgg ggaggaatat gatttacagt ttatttttca actgtgcaaa 1140

ataaccttaa ctgcagacgt tatgacatac atacattcta tgaattccac tattttggag 1200

gactggaatt ttggtctaca acctccccca ggaggcacac tagaagatac ttataggttt 1260

gtaacatccc aggcaattgc ttgtcaaaaa catacacctc cagcacctaa agaagatccc 1320

cttaaaaaat acactttttg ggaagtaaat ttaaaggaaa agttttctgc agacctagat 1380

cagtttcctt taggacgcaa atttttacta caagcaggat tgaaggccaa accaaaattt 1440

acattaggaa aacgaaacgc tacacccacc acctcatcta cctctacaac tgctaaacgc 1500

aaaaaacgta agctgtaa 1518

<210>2

<211>7906

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

actacaataa ttcatgtata aaactaaggg cgtaaccgaa atcggttgaa ccgaaaccgg 60

ttagtataaa agcagacatt ttatgcacca aaagagaact gcaatgtttc aggacccaca 120

ggagcgaccc agaaagttac cacatttatg cacagagctg caaacaacta tacatgatat 180

aatattagaa tgtgtgtact gcaagcaaca gttactgcga cgtgaggtat atgactttgc 240

ttttcgggat ttatgcatag tatatagaga tgggaatcca tatgcagtgt gtgataaatg 300

tttaaagttt tattctaaaa ttagtgagta tagatattat tgttatagtg tgtatggaac 360

aacattagaa cagcaataca acaaaccgtt gtgtgatttg ttaattaggt gtattaactg 420

tcaaaagcca ctatgtcctg aagaaaagca aagacatctg gacaaaaagc aaagattcca 480

taatataagg ggtcggtgga ccggtcgatg tatgtcttgt tgcagatcat cgagaacacg 540

tagagaaacc cagctgtaat catgcatgga gatacaccta cattgcatga atatatgtta 600

gatttgcaac cagagacaac tgatctctac tgttatgagc aattaaatga cagctcagag 660

gaggaggatg aaatagatgg tccagctgga caagcagaac cggacagagc ccattacaat 720

attgtaacct tctgttgcaa gtgtgactct acgcttcggt tgtgcgtaca aagcacacac 780

gtagacatcc gtacgttgga agacctgtta atgggcacac taggaattgt gtgccccatc 840

tgttctcaga aaccataatc taccatggct gatcctgcag gtaccaatgg ggaagagggt 900

acgggatgta atggatggtt ctatgtagag gctgtagtgg aaaaaaaaac aggggatgcc 960

atatcagatg acgagaacga aaatgacagt gatacaggtg aagatttggt agattttata 1020

gtaaatgata atgattattt gacacaggca gaaacagaga cagcacatgc gttgtttact 1080

gcacaggaag caaaagaaca tagagatgca gtacaggttc taaaacgaaa gtatttgggt 1140

agtccactta gtgatattag tgaatgtgta gacaataata ttagtcctag attaaaagcc 1200

atatgtatag aaaaacaaag tagagctgca aaaaggagat tatttgaaag cgaagacagc 1260

gggtatggca atactgaagt ggaaactcag cagatgttac aggtagaagg gcgccatgag 1320

actgaaacac catgtagtca gtatagtggt ggaagtgggg gtggtagcag tcagtatagt 1380

agtggaagtg ggggagaggg tgttagtgaa agacacacta tatgcgaaac accacttaca 1440

aatattttaa atgtactaaa aactagtaat gcaaaggcag caatgctagc aaaatttaaa 1500

gagttatacg gggtgagttt tacagaatta gtaagaccat ttaaaagtaa taaatcaacg 1560

tgttgcgatt ggtgtattgc tgcattcgga cttacaccca gtatagctga cagtataaaa 1620

acattattac aacaatattg tttatattta cacattcaaa gtttagcgtg ttcatgggga 1680

atggttgtgt tactattagt aagatataaa tgtggaaaaa atagagaaac aattgaaaaa 1740

ttgatgtcta aactattatg tgtgtctcca atgtgtatga tgatagagcc tccaaaattg 1800

cgtagtacag cagcagcatt atattggtat aaaacaggta tgtcaaatat tagtgcagtg 1860

tatggagaca cgccagaatg gatacaaaga caaacagtat tacaacatag ttttaatgat 1920

tgtacatttg aattatcaca gatggtacaa tgggcctacg ataatgacat agtagacgat 1980

agtgaaattg catataaata tgcacaattg gcagacacta atagtaatgc aagtgccttt 2040

ttaaaaggta attcacaggc aaaaattgta aaggattgtg caacaatgtg tagacattat 2100

aaacgagcag aaaaaaaaca aatgagtatg agtcaatgga taaaatatag atgtgatagg 2160

gtagatgatg gaggtgattg gaagcaaatt gttatgtttt taaggtatca aggtgtagac 2220

tttatgtcat ttttaagtgc attaaaaaaa tttctgcaag gtatacctaa aaaaaattgc 2280

atattattat atggtgcagc taacacaggt aaatcattat ttggtatgag tttaatgaaa 2340

ttcttgcaag ggtctgtaat atgttttgta aattctaaaa gccatttttg gttacaacca 2400

ttagcagatg ccaaaatagg tatgttagat gatgctacag tgccctgttg gaactacata 2460

gatgacaatt taagaaatgc attggatgga aatttagttt ctatggatgt aaagcataga 2520

ccattggtac aactaaaatg ccctccatta ttaattacat ctaacattaa tgctggtaca 2580

gattccaggt ggccttattt acataatcga ttggtggtgt ttacatttcc aaatgagttt 2640

ccatttgaca aaaacggaaa tccagtgtat gagcttaatg ataagaactg gaaatccttt 2700

ttctcaagga cgtggtccag attaagtttg cacgaggacg aggacaagga aaacgatgga 2760

gactctttgc caacgtttaa atgtgtgtca ggacaaaata ctaacacatt atgaaaatga 2820

tagtacagac ctacgtgacc atatagacta ttggaaacaa atgcgcctag aatgtgctat 2880

ttattacaag gccagagaaa tgggatttaa acatattaac caccaggtgg tgccaacact 2940

ggctgtatca aagaataaag cattacaagc aattgaactg caactaacgt tagaaacaat 3000

atataactca caatatagta atgaaaagtg gacattacaa gacgttagcc ttgaagtgta 3060

tttaactgca ccaacaggat gtataaaaaa acatggatat acagtggaag tgcagtttga 3120

tggagacata tgcaatacaa tgcattatac aaactggaag catatatata tttgtgaaga 3180

cacatcagta actgtggtag agggtcaagt tgactattat ggtatatatt atgttcatga 3240

aggaatacaa acatattttg tgcagtttaa agatgatgca gaaaaatata gtaaaaataa 3300

agtatgggaa gttcatgcgg gtggtcaggt aatattatgt cctacatctg tgtttagcag 3360

cgacgaagta tcctctgctg aaattactag gcagcacttg gccaaccact ccgccgcgac 3420

ccataccaaa gccgtcgcct tgggcaccaa agaaacacag acgactatcc agcgaccaag 3480

atcagagcca gacaccggaa acccctgcca caccaacaag ttgttgcaca gagactccgt 3540

ggacagtgct ccaatcctca ctgcagttaa cagctcacac aaaggacgga ttaactgtaa 3600

tagtaacact acacccatag tacatttaaa aggtgatgct aatactttaa aatgtttaag 3660

atacagattt aaaaagcatt gtaaattgta tacagcagtg tcgtccacat ggcattggac3720

aggacataat gtaaaacata aaagtgcaat tgttacactt acatatgata gtgaatgtca 3780

acgtgaacaa tttttgtctc aagtgaaaat accaaaaact attacagtgt ctactggatt 3840

tatgtctata tgacaaacct tgatactgca tccacaacat tactggcgtg ctttttgctt 3900

tgcttttgtg tgcttttgtg tgtctgccta ttaatacgtc cgctgctttt gtctgtgtct 3960

acatacacat cattaatact attggtatta gtattgtgga taacagcagc ctctgcattt 4020

aggtgtttta ttgtatatat tgtatttgtt tatataccat tatttttaat acatacacat 4080

gcacgcttct taattacata atgtatatgt acataatgta attgttacat ataattgttg 4140

tataccataa attactgatt ttttttttta tttttatttt atatatagtt ttttttctta 4200

tttgtgtgtt ttttaataaa ctgttattac ttaacaatgc gacacaaacg ttctgcaaaa 4260

cgcacaaaac gtgcatcggc cacccaactt tataaaacat gcaaacaggc aggtacatgt 4320

ccacctgaca ttatacctaa ggttgaaggc aaaactattg ctgatcaaat attacaatat 4380

ggaagtatgg gtgtattttt tggtgggtta ggaattggaa cagggtctgg tacaggcgga 4440

cgcactgggt acattccatt aggaacaagg cctcccacag ctacggatac acttgctcct 4500

gtaagacccc ctttaacagt agatcctgtg ggcccttctg atccttctat agtttcttta 4560

gtggaagaaa ctagttttat tgatgctggt gcaccaaccc ctgtaccttc cattccccca 4620

gatgtatcag gatttagtat tacaacttca actgatacca cacctgctat attagatatt 4680

aataatactg ttactactgt tactacacat aataatccca cttttactga cccatctgta 4740

ttgcagcctc caacacctgc agaaactgga gggcatttta cactttcatc atccactatt 4800

agtacacata attatgaaga aattcctatg gatacattta ttgttagcac aaaccctaac 4860

acagtaacta gtagcacacc cataccgggg tctcgcccag tggcacgcct aggattatat 4920

agtcgcacaa cacaacaagt taaggttgtg gaccctgctt ttgtaaccgc tcccactaaa 4980

cttattacat atgataatcc tgcatatgaa ggtatagatg tggataatac attttatttt 5040

cctagtaatg ataatagtat taatatagct ccagatcctg actttttgga tatagttgct 5100

ttacataggc cagcattaac ctctaggcgt actggcatta gatacagtag aattggtaat 5160

aaacaaacac tacgtactcg tagtggaaaa tctataggtg ctaaggtaca ttattattat 5220

gatttaagta ctattaatcc tgcagaagaa atagaattac aaactataac accttctaca 5280

tatacaacca cttcccatgc agcctcacca acttctatta ataatggatt atatgatatt 5340

tatgcagatg actttattac agatactgtt acaaccccag taccagctat accctctaca 5400

tccttatcag gttatattcc tgcaaataca acaattcctt ttggtggtgc atacaatatt 5460

cctttagtat caggacctga tatacctatt aatacaactg accaaactcc ttcattaatt 5520

cctatagttc cagggtctcc acaatataca attattgctg atggaggtga cttttattta 5580

catcctagtt attacatgtt acgaaaacga cgtaaacgtt taccatattt tttttcagat 5640

gtctctttgg ctgcctagtg aggccactgt ctacttgcct cctgtcccag tatctaaagt 5700

tgtaagcacg gatgaatatg ttgcacgcac aaacatatat tatcatgcag gaacatccag 5760

actacttgca gttggacatc cctattttcc tattaaaaaa cctaacaata acaaaatatt 5820

agttcctaaa gtatcaggat tacaatacag ggtatttaga atatatttac ctgaccccaa 5880

taagtttggt tttcctgaca cctcatttta caatccagat acacagcggc tggtttgggc 5940

ctgtgtaggt gttgaggtag gtcgtggtca gccattaggt gtgggcatta gtggccatcc 6000

tttattaaat aaattggatg acacagaaaa tgctagtgct tatgcagcaa atgcaggtgt 6060

ggataataga gaatgtatat ctatggatta caaacaaaca caattgtgtt taattggttg 6120

caaaccacct ataggggaac actggggcaa aggatcccca tgtaacaatg ttgcagtaac 6180

tccaggtgat tgtccaccat tagagttaat aaacacagtt attcaggatg gtgatatggt 6240

tgataccggc tttggtgcta tggactttac tacattacag gctaacaaaa gtgaagttcc 6300

actggatatt tgtacatcta tttgcaaata tccagattat attaaaatgg tgtcagaacc 6360

atatggcgac agcctatttt tttatttacg aagggaacaa atgtttgtta gacatttatt 6420

taatagggct ggtgctgttg gtgaaaatgt accagacgat ttatacatta aaggctctgg 6480

gtctactgca aatttagcca gttcaaatta ttttcctaca cctagtggtt ctatggttac 6540

ctctgatgcc caaatattta ataaacctta ttggttacaa cgagcacagg gccacaataa 6600

tggcatttgt tggggtaacc aactatttgt tactgttgtt gatactacac gcagtacaaa 6660

tatgtcatta tgtgctgcca tatctacttc agaacctaca tataaaaata ctaactttaa 6720

agagtaccta cgacatgggg aggaatatga tttacagttt atttttcaac tgtgcaaaat 6780

aaccttaact gcagacgtta tgtcatacat acattctatg aattccacta ttttggagga 6840

ctggaatttt ggtttacaac ctcctccagg aggcacacta gaagatactt ataggtttgt 6900

aacatcccag gcaattgctt gtcaaaaaca tacacctcca gcacctaaag aagatcccct 6960

taaaaaatat actttttggg aagtaaattt aaaagaaaag ttttctgcag acctagatca 7020

gtttccttta ggacgcaaat ttttactaca agcaggattt aaggccaaac caaaatttac 7080

attaggaaaa cgaaaagcta cacccaccac ctcatctacc tctacaactg ctaaacgcaa 7140

aaaacgtaag ctgtaagtat tgtatgtatg ttgaattagt gttgtttgtt gtttatatgt 7200

ttgtatgtgc ttgtatgtgc ttgtaaatat tacgttgtat gtgtgtttgt atgtatggta 7260

taataaacac gtgtgtatgt gtttttaaat gcttgtgtaa ctattgtgtc atgcaacata 7320

aataaactta ttgtttcatc acctactaat tgtgttgtgg ttattcattg tatataaact 7380

atatttgcta cattttgttt ttgttttata tatactatat tttgtagcgc cagcggccat 7440

tttgtagctt caaccgaatt cggttgcatg ctttttggca caaactgtat ttttttaaat 7500

agttctatgt cagcaactat agtttaaact tgtacgtttc ctgcttgcca tgcgtgccaa 7560

atccctgttt tcctgacctg cactgcttgc caaccattcc attgtttttt acactgcact 7620

atgtgcaact actgaatcac tatgtacatt gtgtcatata aaataaatta ctatgcgcca 7680

acgccttaaa taccgctgtt aggcacatat ttttggcttg ttttaactca cctaattgca 7740

tagttggcat aaggtttaaa cttttaaggc caactaaatg tcaccttagt tcatacatga 7800

actgtgtaaa ggttagtcat acattgttca tttgtaaaac tgcacatggg tgtgtgcaaa 7860

ccgttttggg ttacacattt acaagaaact tatataataa tactaa 7906

<210>3

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cggaattcaa atgtgcctgt atacacgggt c 31

<210>4

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cgcggatccg cgcacacata ttacttcctg g 31

<210>5

<211>81

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ctttgctttt gtgtgctttt gtgtgtccca catttatgca cagagctgca aacattagat 60

ttgcaaccag agacaactga t 81

<210>6

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Phe Leu Leu Cys Phe Cys Val Leu Leu

1 5

<210>7

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Lys Leu Pro Gln Leu Cys Thr Glu Leu

1 5

<210>8

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Tyr Met Leu Asp Leu Gln Pro Glu Thr

1 5

Claims (9)

1. A group of immune antigen peptide coding nucleic acid sequences is characterized by consisting of three nucleic acid fragments which respectively code E5: F LL CFCV LL, E6: K L PQ L CTE L and E7: YM L D L QPET.

2. Use of the coding nucleic acid sequence of the three peptide fragments HPV16E5, E6 and E7 as claimed in claim 1 in the preparation of preparations for tumor and its prevention and treatment in research of HPV16 related diseases.

The cervical cancer vaccine preparation prepared by combining HPV 16L 1 protein coded by SEQ ID NO. 1 and E5, E6 and E7 antigenic peptides of claim 1.

4. A method for preparing chimeric virus-like particles cV L Ps is characterized in that three nucleic acid fragments as described in claim 1 and HPV 16L 1 as shown in SEQ ID NO: 1 are expressed by an insect cell-baculovirus expression system, and HPV 16L 1+ "E5E 6E 7" chimeric virus-like particles cV L Ps are finally obtained through construction, verification, amplification and purification respectively, wherein packaging amplification HPV 16L 1V L P is used as a control research.

5. The method for preparing cV L Ps according to claim 4, wherein a Bac to Bac baculovirus expression system is used, HPV 4831 + "E5E 6E 7" gene is cloned by a PCR method, after sequencing identification, the gene is connected with linearized pFastHTb to pFastHTb-HPV 16L + "E5E 6E 7", pFastHTb-HPV 16L + "E5E 6E 7" is subjected to transposition recombination with DH10BAC competent bacteria, resistant and blue-white spot screening recombination Bacmid-HPV 16L + "E5E 6E 7" clone is used, mid-HPV 16L 1+ "E5E 6E 7" is extracted to transfect insect cells Sf-9 to obtain infectious viruses, Sf-9 cells are infected by the viruses to carry out protein expression and amplification, RT-PCR and Western blot are used for identification, and cV 7 expressing HPV16 type E3 + "E5E 6E 7372" is screened.

6. The method of claim 5, wherein the upstream and downstream primer sequences of HPV 16L 1 are L1-P1: 5'CGGAATTCAAATGTGCCTGTATACACGGGTC 3'; L1-P2: 5'CGCGGATCCGCGCACACATATTACTTCCTGG3', respectively.

7. The chimeric virus-like particles cV L Ps prepared by the method of claim 4 or 5.

8. The use of the cV L Ps prepared by the method for preparing the chimeric virus-like particles cV L Ps according to claim 4 or 5 in preparing a cervical cancer vaccine with both prevention and treatment effects.

9. Use of the cV L Ps prepared by the method for preparing the chimeric virus-like particles cV L Ps according to claim 4 or 5 in preparing a reagent for the pathogenesis and immunotherapy of cervical cancer.

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