CN114164215A - Anti-trichina small heat shock protein egg yolk antibody and preparation method and application thereof - Google Patents
Anti-trichina small heat shock protein egg yolk antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a chicken egg yolk antibody of anti-trichina small heat shock proteins (sHSPs) and a preparation method thereof, and also discloses medical application of the egg yolk antibody in tumor resistance; has the characteristics of enhancing the immunity of animals, being nontoxic and the like, can be used as an oral preparation medicament for treating tumors, and can also be prepared into oral liquid and the like as food for preventing tumors; the egg yolk antibody of poultry such as chicken and the like can be orally taken, is convenient to use, has no toxicity, is low in cost and safe, and is convenient for large-scale industrial production.
Description
The technical field is as follows:
the invention provides a trichina-resistant small heat shock protein egg yolk antibody, relates to an egg yolk antibody for resisting trichina and Lewis lung cancer related gene protein antigens sHSPs, and particularly discloses a chicken egg yolk antibody for resisting trichina and Lewis lung cancer related gene protein antigens sHSPs and a preparation method thereof; simultaneously, also provides the medical application of the yolk antibody in tumor resistance, belonging to the technical field of biological medicine.
Background art:
trichina (A)Trichinella spiralis) Is a zoonotic parasite in which adults live in the small intestine of mammals and larvae live in muscle tissue. At present, researches show that trichina can cause trichinosis, and trichinosis infection can also improve the anti-tumor capability of a host. At present, some related researches on the polyclonal antibody and monoclonal antibody of trichina and cancer cell related gene protein antigen have been reported, and it is found that the polyclonal antibody and monoclonal antibody of trichina and tumor related gene protein antigen have an inhibiting effect on tumor cells, and mouse serum and monoclonal antibodies prepared aiming at the related protein antigens have a certain anti-tumor effect in mouse animal experiments, but the used antibodies are all used by injection, so that the clinical popularization and application are difficult. There is no report of using fowl egg yolk antibody of trichina and tumor-related gene protein antigen for oral administration for preventing and treating cancer.
The invention content is as follows:
the invention provides a Small heat shock protein (sHSPs) chicken egg yolk antibody against trichina and a preparation method thereof, and has the advantages of convenient use, low cost, safety, no toxicity, convenience for large-scale industrial production and the like.
The invention further discloses the application of the egg yolk antibodies of poultry such as trichina small heat shock protein (sHSPs) chicken and the like, has the characteristics of enhancing the immunity of animals, and being non-toxic and harmless, can be used as an oral preparation for treating tumors, and can also be prepared into oral liquid and the like for preventing tumors.
The gene sequence of the trichina and Lewis lung cancer related gene protein antigen sHSPs is shown in SEQ No. 1.
The trichina and Lewis lung cancer related protein gene sHSPs disclosed by the invention have the following characteristics:
the gene has the accession number of DQ986457.1, codes small heat shock proteins (sHSPs), has 498bp open reading frames, codes 165 amino acids, has an isoelectric point of 6.62 and a molecular weight of about 19 KDa.
The invention relates to a chicken egg yolk antibody of anti-trichina small heat shock protein (sHSPs), which has the following characteristics:
1. SDS-PAGE detection shows that the heavy chain of the yolk antibody is about 68KDa, and the light chain is about 38KDa;
2. ELISA and Western blot detection show that the yolk antibody can react with the sHSPs.
The invention relates to a preparation method of a trichina small heat shock protein (sHSPs) chicken egg yolk antibody, which comprises the following steps:
1) designing a pair of primers according to the specific gene sequence of the trichina small heat shock protein;
an upstream primer: 5'-ATGGCCTTGACTGCGTGGTATAATC-3', respectively;
a downstream primer: 5'-TCATCTCTTCTGAGCAGCCGGAGC-3', respectively;
2) PCR amplification is carried out on the sHSPs gene by taking trichina muscle larva cDNA as a template, and the sHSPs gene is connected with a cloning vector pMD-18T (pMD-18T-sHSPs);
3) after sequencing, pMD-18T-sHSPs and empty vector pET-32a (+) are respectively cut by double enzymes, T4 DNA ligase is used for connection, and BL-21 (DE 3) is transformed after pMD-18T-sHSPs and pET-32a (+) are recovered by glue;
4) inducing expression of the recombinant bacteria with correct sequencing by IPTG, collecting the bacteria, carrying out ultrasonic crushing, taking the supernatant, and purifying by a nickel column affinity chromatography; and (3) immunizing the Roman hen after measuring the concentration by using a BCA method, collecting eggs, separating egg yolk, and separating and purifying the egg yolk antibody by using a gradient PEG6000 method.
The invention relates to a medical application of a trichina small heat shock protein (sHSPs) chicken egg yolk antibody in a medicine for preventing and treating tumors.
The invention relates to a medical application of a trichina small heat shock protein (sHSPs) chicken egg yolk antibody in a medicine for preventing and treating lung cancer.
The pharmacological tests prove that the anti-trichina sHSPs egg yolk antibody has obvious effect of inhibiting tumor growth, and can be applied as a preparation for preventing and treating tumors:
c57 mice were inoculated with Lewis lung carcinoma cells 12d and randomized into 2 groups of 6 cells each. Gavage treatment was performed at a dose of 100 μ g per group, where the pre-immune yolk antibody group: 100 mu g; anti-sHSPs egg yolk antibody group: 100 μ g. Serial treatment for 15d, 3d after the last treatment, serum was isolated for cytokine detection, and mice were euthanized, tumor tissue was isolated, and volume and weight were measured. And analyzed with SPSS 23.0 software. Meanwhile, taking partial mouse heart, liver, spleen, lung, kidney and other tissues to fix with formalin and staining by HE. The tumor inhibition rate was calculated by volume and weight analysis of tumors in each group of mice, and it was found that the inhibition rate of the yolk antibody against sHSPs was 44.80%. Cytokine assays found that anti-sHSPs yolk antibodies significantly increased IFN-. gamma.IL-2 and TNF-. alpha.levels. HE staining results of tissues and organs of each group of mice show that the anti-sHSPs egg yolk antibody does not cause pathological damage.
The invention has the positive effects that:
provides a chicken egg yolk antibody of anti-trichina small heat shock protein (sHSPs), and also discloses the medical application of the egg yolk antibody in tumor resistance; has the characteristics of enhancing the immunity of animals, being nontoxic and the like, can be used as an oral preparation medicament for treating tumors, and can also be prepared into oral liquid and the like as food for preventing tumors; the egg yolk antibody of poultry such as chicken and the like can be orally taken, is convenient to use, has no toxicity, is low in cost and safe, and is convenient for large-scale industrial production.
Description of the drawings:
FIG. 1 is an SDS-PAGE analysis electrophoresis of the trichinella sHSPs (M. protein marker 1. purified sHSPS);
FIG. 2 is a Western blot analysis electrophoresis chart of the Trichinella spiralis sHSPs (M. protein marker 1. purified sHSPS);
FIG. 3 is an SDS-PAGE analysis electrophoresis of the anti-sHSPs yolk antibody of the invention (M. protein marker 1.anti-sHSPS IgY 2. pre-immune control);
FIG. 4 is a Western blot analysis electrophoretogram of the anti-sHSPs egg yolk antibody (upper graph M. protein marker 1.2.3.anti-sHSPS IgY lower graph and pre-immune control);
FIG. 5 is a graph showing HE staining of tissues and organs of various groups of mice according to the present invention.
The specific implementation mode is as follows:
for the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example 1:
trichina and Lewis lung cancer related antigen gene sHSPs expression
Designing a primer according to a trichina small heat shock protein (sHSPs) specific gene sequence (DQ 986457.1), wherein the nucleotide sequence of the primer is as follows:
an upstream primer: 5'-ATGGCCTTGACTGCGTGGTATAATC-3', respectively;
a downstream primer: 5'-TCATCTCTTCTGAGCAGCCGGAGC-3', respectively;
the restriction enzyme sites are respectively: GGATCC (BamHI); GTCGAC (SalI); carrying out PCR amplification on sHSPs gene by taking trichina muscle larva cDNA as a template, connecting the sHSPs gene with a cloning vector pMD-18T to be named as pMD-18T-sHSPs, carrying out double digestion on the pMD-18T-sHSPs and an empty vector pET-32a (+) after sequencing verification, respectively connecting the pMD-18T-sHSPs and the pET-32a (+) recovered by using T4 DNA ligase, finally converting BL-21 (DE 3), carrying out IPTG induced expression on recombinant bacteria with correct sequencing, collecting bacteria, carrying out ultrasonic crushing, and taking supernatant for later use;
purification of trichina and Lewis lung cancer related antigen sHSPs
Collecting the above supernatant, purifying by nickel column affinity chromatography, determining concentration by BCA method, sampling 50 μ l of purified sample, and performing SDS-PAGE and Western blot identification to obtain results shown in FIGS. 1 and 2; storing other samples at-20 ℃;
extraction and purification of sHSPs-resistant egg yolk antibody
The trichina sHSPs are mixed and emulsified with equal volume of Freund's complete adjuvant at the dosage of 500 mug/single, and then the lateral thigh and the posterior chest are immunized in multiple points. After the first immunization for 14 days, 250 mu g of sHSPs are mixed with equal volume of Freund's incomplete adjuvant to strengthen the immunization, and the strengthening immunization is carried out for 3 times at intervals of 10 days; collecting eggs 7 days after the first time, and storing at 4 ℃;
the eggs were wiped on the surface with 75% alcohol. Breaking egg shell to separate egg yolk, puncturing egg yolk membrane, and collecting egg yolk. Mixing yolk and PBS (5 times volume of the yolk) and standing at 4 ℃ for 30min, adding 3.5% PEG6000, standing for 10min, centrifuging at 4 ℃ and 8000r/min for 30min, taking the supernatant, filtering with gauze, adding 8.5% PEG6000, standing for 10min, centrifuging at 4 ℃ and 8000r/min for 30min, discarding the supernatant, dissolving the precipitate with PBS, adding 12% PEG6000, standing for 10min, centrifuging at 4 ℃ and 8000r/min for 30min, dissolving the precipitate with PBS, obtaining IgY, desalting and concentrating with a 3KDa ultrafiltration tube. The concentration was measured by BCA method and stored at-20 ℃.
Test example 1
Potency and identification of anti-sHSPs egg yolk antibodies
The sHSPs prepared in example 1 were coated at 1. mu.g/well, washed 3 times with 200. mu.l PBST overnight at 4 ℃, blocked for 2h at 37 ℃ using 100. mu.l PBST-solubilized 5% skim milk powder, washed, incubated with diluted IgY at 37 ℃ for 2h, washed, added with 100. mu.l rabbit anti-chicken IgG (1: 500), incubated for 45min at 37 ℃, washed, added with 100. mu.l TMB color former, incubated for 15min, added with 100. mu.l stop buffer, and read the OD450 value with a microplate reader. As a result, the titer of the anti-sHSPs egg yolk antibody after the fourth immunization can reach 1: 102400 (table 1):
TABLE 1 anti-sHSPs egg yolk antibody titer (OD 450)
50. mu.l of the preimmune IgY and the anti-sHSPs IgY prepared in example 1 were sampled and subjected to SDS-PAGE and Coomassie Brilliant blue staining, respectively, as shown in FIG. 3.
Mu.g of the sHSPs prepared in example 1 were subjected to SDS-PAGE and transferred onto PVDF membrane, blocked with TBST-solubilized 5% skim milk powder at room temperature for 2h, incubated overnight at 4 ℃ with the preimmune IgY and the anti-sHSPs IgY (1: 500), respectively, washed 3 times with TBST, then incubated with rabbit anti-chicken IgG (1: 500) at room temperature for 45min, washed for 2h, and ECL luminophore was dropped onto the membrane and visualized with a chemiluminescence gel imager, see FIG. 4.
Test example 2
anti-sHSPs egg yolk antibody anti-tumor assay
2 x 10 to6Lewis lung carcinoma cells were inoculated into C57 mice 12d and then randomized into 2 groups of 6 cells each. Gavage treatment was performed at a dose of 100 μ g per group, where the pre-immune yolk antibody group: 0.1ml (100. mu.g); anti-sHSPs egg yolk antibody group: 0.1 ml. Treating with yolk antibody for 15 days, collecting blood 3 days after the last time, and separating serum. Mice were sacrificed post anesthesia, tumor tissue was isolated, weighed, and volume was measured. And taking organ tissues of the heart, the liver, the spleen, the lung and the kidney of the mouse to perform formalin fixation for later use. All data were analyzed using SPSS 23.0 software. The test results are shown in table 2:
TABLE 2 tumor volume and weight in mice of each group
| Group of | Number of | Tumor volume (mm 3)/inhibition ratio | Tumor (g)/inhibition ratio |
| Preimmune IgY | 6 | 386.47±27.40（5.33%） | 1.45±0.07（5.84%） |
| anti-sHSPs IgY | 6 | 225.33±30.56***（44.80%） | 0.86±0.05***（44.16%） |
*P<0.05，** P <0.01， ***P <0.001。
And (4) conclusion: through statistical analysis of the weight and volume of the tumors of each group of mice, the tumor inhibition rate of each group is calculated, and the result shows that the sHSPs-resistant yolk antibody can obviously inhibit the tumor growth, the tumor inhibition rate is 44.80 percent, and the difference is significant compared with a yolk antibody control group before immunization (P is less than 0.001);
test example 3
Evaluation of Effect of anti-sHSPs egg yolk antibody on cytokine and safety
The mouse serum obtained in test example 2 was detected using a commercial cytokine detection kit. As a result, the egg yolk antibody resisting the sHSPs increases the expression level of IFN-gamma, IL-2 and TNF-alpha; the results are shown in Table 3.
TABLE 3 Effect of anti-sHSPs egg yolk antibodies on tumor-bearing mouse cytokines
| Group of | IFN-γ | TNF-α | IL-2 | IL-4 | IL-10 |
| Preimmune IgY | 44.83±7.81 | 69.57±5.22 | 241.38±8.29 | 180.56±9.22 | 166.71±8.24 |
| anti-sHSPs IgY | 57.64±7.72** | 81.2±3.84* | 259.50±7.02* | 177.36±6.72 | 169.13±10.97 |
*P<0.05， ** P <0.01，***P <0.001。
And (4) conclusion: the HE staining of formalin-fixed tissues such as heart, liver, spleen, lung and kidney of each group of mice obtained in the experimental example 2 was performed by a conventional method, and pathological damage effects of yolk antibodies on tissues and organs of the body were observed, and as a result, it was found that no significant pathological damage was found in the IgY and sHSPs yolk antibody groups before immunization, i.e., the prepared anti-trichina sHSPs yolk antibodies did not cause pathological damage to the body, as shown in fig. 5.
Sequence listing
<110> Jilin university
<120> a trichina resistant small heat shock protein egg yolk antibody and preparation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 498
<212> DNA
<213> Trichinella spiralis
<400> 1
atggccttga ctgcgtggta taatccgtgg gttgatgatc cattttggcc gtcgtctcga 60
ccggctcgct tcttcgagga tctgagccgc atgatacgca gctttgatga catgtatcga 120
ggtttgccga accagatgaa cgtgccaatg aatgagctgt cgatgtgcgg aaatgcatcc 180
gaggtggtga acaacgacaa caagtttgaa gtttcactcg acgtgaagca tttcaaacct 240
gaagagctga ctgtgaaaac taccgacaat cgtctcgtca tcaccggcaa gcatgaggag 300
aagcaagatg agcatggctt tgtcaaacgc gagttttctc gttcttacta tctaccacaa 360
ggcgttaagc cggatcagtt tgtctccaac ttgggtccgg atggaaagtt ggttattaca 420
gcaccgaagc aggcgattga aggcagcaac gaacgaaaaa ttccaatcac cgccgctccg 480
gctgctcaga agagatga 498
<210> 2
<211> 25
<212> DNA
<213> Trichinella spiralis
<400> 2
atggccttga ctgcgtggta taatc 25
<210> 3
<211> 24
<212> DNA
<213> Trichinella spiralis
<400> 3
tcatctcttc tgagcagccg gagc 24
Claims (6)
1.A gene sequence of trichina and Lewis lung cancer related gene protein antigen sHSPs is shown in SEQ No. 1.
2. The trichina and Lewis lung cancer related protein gene sHSPs have the following characteristics:
the gene has the accession number of DQ986457.1, codes small heat shock proteins (sHSPs), has 498bp open reading frames, codes 165 amino acids, has an isoelectric point of 6.62 and a molecular weight of about 19 KDa.
3.An anti-trichina small heat shock protein (sHSPs) chicken egg yolk antibody characterized by:
1) SDS-PAGE detection shows that the heavy chain of the yolk antibody is about 68KDa, and the light chain is about 38KDa;
2) ELISA and Western blot detection show that the yolk antibody can react with the sHSPs.
4. The method for preparing chicken egg yolk antibody against small heat shock proteins (sHSPs) of trichina of claim 3, which comprises the following steps:
1) designing a pair of primers according to the specific gene sequence of the trichina small heat shock protein;
an upstream primer: 5'-ATGGCCTTGACTGCGTGGTATAATC-3', respectively;
a downstream primer: 5'-TCATCTCTTCTGAGCAGCCGGAGC-3', respectively;
2) PCR amplification is carried out on the sHSPs gene by taking trichina muscle larva cDNA as a template, and the sHSPs gene is connected with a cloning vector pMD-18T (pMD-18T-sHSPs);
3) after sequencing, pMD-18T-sHSPs and empty vector pET-32a (+) are respectively cut by double enzymes, T4 DNA ligase is used for connection, and BL-21 (DE 3) is transformed after pMD-18T-sHSPs and pET-32a (+) are recovered by glue;
4) inducing expression of the recombinant bacteria with correct sequencing by IPTG, collecting the bacteria, carrying out ultrasonic crushing, taking the supernatant, and purifying by a nickel column affinity chromatography; and (3) immunizing the Roman hen after measuring the concentration by using a BCA method, collecting eggs, separating egg yolk, and separating and purifying the egg yolk antibody by using a gradient PEG6000 method.
5. The medical use of the trichina small heat shock protein (sHSPs) chicken egg yolk antibody as claimed in claim 2 in a medicine for preventing and treating tumor.
6. The medical use of the trichina small heat shock protein (sHSPs) chicken egg yolk antibody as claimed in claim 2 in a medicament for preventing and treating lung cancer.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215170A (en) * | 2021-05-30 | 2021-08-06 | 吉林大学 | sHSPs recombined invasive lactobacillus plantarum live vector DNA vaccine |
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2020
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| JP2004238401A (en) * | 2004-05-06 | 2004-08-26 | Kotoku Seibutsu Kagi Kofun Yugenkoshi | Separation and purification method of egg yolk antibody of bird and use of the obtained egg yolk antibody |
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| WO2021112174A1 (en) * | 2019-12-03 | 2021-06-10 | 国立大学法人東海国立大学機構 | Optical antimicrobial therapeutic method |
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| 岳涛涛: "旋毛虫与Lewis肺癌相关抗原基因sHSPs重组侵入型植物乳杆菌抗肿瘤效应研究" * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113215170A (en) * | 2021-05-30 | 2021-08-06 | 吉林大学 | sHSPs recombined invasive lactobacillus plantarum live vector DNA vaccine |
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