CN101658667B - Cryptosporidium parvum divalent protein vaccine and preparation method thereof - Google Patents
Cryptosporidium parvum divalent protein vaccine and preparation method thereof Download PDFInfo
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- CN101658667B CN101658667B CN2009100664798A CN200910066479A CN101658667B CN 101658667 B CN101658667 B CN 101658667B CN 2009100664798 A CN2009100664798 A CN 2009100664798A CN 200910066479 A CN200910066479 A CN 200910066479A CN 101658667 B CN101658667 B CN 101658667B
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Abstract
The invention provides a cryptosporidium parvum divalent protein vaccine, which is characterized in that prokaryotic expression vector pET28a is taken as a carrier, and two cryptosporidium parvum protective antigen genes are inserted in series to the multiple cloning sites of the prokaryotic expression pET28a, thus obtaining the cryptosporidium parvum divalent protein vaccine. The vaccine selects two advantageous protective antigens, combines the advantages of the two antigens, and can induce stronger functions of cellular immunity and humoral immunity, thus achieving the purposes of preventing cryptosporidium parvum disease of animal better.
Description
Technical field
The invention provides a kind of cryptosporidium parvum divalent protein vaccine, be used for the prevention and the treatment of cryptosporidiosis, also provide simultaneously this vaccine method for preparing, belong to infectious disease vaccine production technical field.
Background technology
Cryptosporidium (Cryptosporidium) is a kind of parasitic protozoa of infecting both domestic animals and human, and main parasitic is claimed cryptosporidiosis by the disease that it causes in the epithelial cell of people and the mucosas such as respiratory tract of other mammiferous gastrointestinal tract, birds.Cryptosporidiosis distributes extensively, and the human poultry infection leads height, and harm is serious, is principal character with serious watery diarrhea clinically.The Cryptosporidium spp rate can reach 10%～50% in aids patient, is one of main lethal factor of aids patient.At all Cryptosporidium kind apoplexy due to endogenous wind, maximum to the human health danger side of body with Cryptosporidum parvum, U.S. CDC is classified Cryptosporidum parvum as the category-B biological weapons.This disease is that the important parasite of medical science and veterinary educational circles control from now on is sick.Though through research for many years; Still there are not effectively preventing medicine and prevention method so far; Screened more than 200 kind of medicine, comprised that all antibiotic, anticoccidial drug and sulfa drugs are used for the treatment of this disease, but do not have specific medicine can play killing action Cryptosporidium.Infection character and the immunologic function of Cryptosporidium are closely related, its state of an illness weight, course of disease length behind the host infection Cryptosporidium, and therapeutic effect possibly depend primarily on modulation on immune status.Therefore, immunologic intervention possibly be a more suitably approach of control cryptosporidiosis.
Summary of the invention
The object of the invention provides a kind of cryptosporidium parvum divalent protein vaccine, is the new generation vaccine of anti-Cryptosporidum parvum, for the prevention and the treatment of cryptosporidiosis positive effect is arranged.
The present invention also provides the method for preparing of this vaccine, is applicable to suitability for industrialized production.
Cryptosporidium parvum divalent protein vaccine of the present invention; It is characterized in that: with prokaryotic expression carrier pET28a is carrier; The series connection of two Cryptosporidum parvum protective antigen genes is inserted into the MCS district of pET28a prokaryotic expression carrier, has obtained cryptosporidium parvum divalent protein vaccine.
The preparation technology of cryptosporidium parvum divalent protein vaccine of the present invention may further comprise the steps:
Read frame design primer according to the opening of Cryptosporidum parvum protective antigen gene sequence and carry out PCR, the TA clone.Two protective antigen gene series connection of gained are cloned into prokaryotic expression carrier pET28a carrier, make up the Cryptosporidum parvum bivalent recombinant protein vaccine.Use the IPTG abduction delivering, product carries out the activity of SDS-PAGE electrophoresis, Western blot analysis expression product.After recombiant protein is purified, carry out the checking of animal protection property test effect.
The concrete preparation technology of two divalent protein vaccines may further comprise the steps:
CP12, CP21 genes of interest are connected with the T carrier respectively; After cloning and sequencing is correct; Carry out the double digestion reaction with BamHI, EcoR1 and EcoR1, Xhol respectively; Reclaim fragment and be connected with the prokaryotic expression carrier pET-28a of Xhol double digestion reaction, make up the pET28-CP12-CP21 recombinant vector through BamHI.
Good effect of the present invention is: selected for use two protective antigen gene series connection to carry out the development of two divalent protein vaccines, to strengthen its immune protective effect.The Cryptosporidum parvum protein vaccine can cause cellular immunization and the humoral immune reaction that body is stronger; And two genes that this research is introduced all are again advantage protective antigen genes; Therefore more intensive immune response can be produced, thereby the sick purpose of better prevention animal Cryptosporidum parvum can be reached.
Description of drawings
Fig. 1: be pET28-CP12-CP21 vector construction sketch map;
Fig. 2: be the SDS-PAGE and the Western blotting evaluation figure of recombiant protein;
Fig. 3: be the protective effect of protein vaccine to the anti-Cryptosporidum parvum infection of immune mouse.
Table 1: be each group mice serum specific antibody IgG titre situation of change
Table 2: be test group and control group mice spleen CD4
+And CD8
+The T lymphocyte quantity
The specific embodiment
The preparation of cryptosporidium parvum divalent protein vaccine
The present invention is an example with Cryptosporidum parvum immunity related protein gene CP12, CP21, carries out the structure of recombinant prokaryotic expression vector.
The preparation process of cryptosporidium parvum divalent protein vaccine:
Design primer and introduce restriction enzyme site with reference to CP12, CP21 gene order open reading frame and prokaryotic expression carrier pET28a physical map.
The CP12 forward primer:
QF1:5 '-CT
GGATCCATGTCAGATGCATCAATA-3 '; Wherein the 5` end contains the BamHI site;
Downstream primer QR1:5 '-GGGGC
GAATTCTATTTGTTCATTCATCTG-3 '; The 5` end contains the EcoRI site.
The CP21 forward primer:
QF2:5 '-CC
GAATTCGGTGGCGGTGGCTCGATGTCTAAAAGAGCAT-3 ' wherein 5` end contains EcoRI site and Linker sequence;
Downstream primer QR2:5 '-CTGGCT
CTCGAGCTATTCATCTGTTTGAAC-3 '; The 5` end contains the XhoI site.
(general parameter is: 94 ℃ of 4min to utilize above-mentioned primer sequence to carry out the PCR reaction; 94 ℃ of 45s, 55 ℃ of 50s, 72 ℃ of 1min extend 10min in 72 ℃ after 30 circulations), each PCR purified product clone respectively to the PMD18-T carrier, after PCR and the evaluation of corresponding enzyme action, is sent to biotech firm's order-checking and identifies.Gel reclaims each purpose fragment and is connected with the prokaryotic expression carrier pET28a that carries out double digestion with BamHI and XhoI then; Screen recombiant plasmid after connecting product Transformed E .coli DH5 α competent cell; Carrying out the double digestion reaction with BamHI and XhoI identifies; With identifying that correct plasmid transforms the DE3 competence and carries out double digestion reaction evaluation with BamHI and XhoI, with recombiant plasmid called after pET28-CP12-CP21 (shown in accompanying drawing 1).The recombiant plasmid that builds is used the IPTG abduction delivering, and product carries out the activity that SDS-PAGE electrophoresis, Westernblot are analyzed expression product, and the expressed destination protein that goes out is carried out purification (shown in accompanying drawing 2) through the method for affinity chromatograph.
The purposes of cryptosporidium parvum divalent protein vaccine
One, the purposes of cryptosporidium parvum divalent protein vaccine
With 60 4～6 ages in week; The female cleaning level BALB/c mouse of 18～22g is divided into 3 groups; Every group 20, test group is through the lumbar injection recombiant protein, and immunity is three times altogether; In each 2 weeks at interval, immunity for the first time is with 0.1mL antigenic solution (containing 20 μ g recombiant proteins) and the injection of equal-volume Freund's complete adjuvant emulsifying pneumoretroperitoneum; Inject with 0.1mL antigenic solution (containing 20 μ g recombiant proteins) and equal-volume incomplete Freund emulsifying pneumoretroperitoneum with immunity for the third time for the second time.The Freund adjuvant of adjuvant matched group (Negativecontrol B) lumbar injection and test group equivalent is injected Freund's complete adjuvant for the first time, injects incomplete Freund for the second time and for the third time.Blank group lumbar injection PBS (Negativecontrol A).
Three exempt from back one all oral inoculation Cryptosporidum parvum egg capsules 1 * 10
6Individual/as only to attack worm test, attack to collect every three days behind the worm and respectively organize feces, collect egg capsule with saturated zinc sulfate floatation, deposition is dissolved in a certain amount of water, behind the mixing, to get one and place blood cell counting plate, the Cryptosporidium egg capsule that under low power lens, counts is total.Carry out the detection of body fluid and cellular immune level simultaneously.Tail vein blood before each immunity, separation of serum, detects IgG situation of change in the Mus serum through indirect ELISA method as envelope antigen with Cryptosporidum parvum egg capsule antigen.1 week of back of immunity for the third time, get 4 mice sacrificed by exsanguination at random for every group, get spleen, add 2mL PBS, on 200 mesh sieves, grind, be diluted to cell suspension (10 with PBS
7Individual/mL).Get 100 μ L cell suspension, add the anti-CD4 of FITC labelling
+Anti-CD8 with the PE labelling
+The anti-mouse monoclonal antibody of rabbit, lucifuge effect 40min washes 2 times with the PBS washing liquid then, adds 0.5mL fluorescence and preserves liquid, detects CD4 with flow cytometer
+, CD8
+The lymphocytic quantity of T, and the gained data are carried out statistical analysis with SPSS software.
The immune protective result of the test shows that the test group immune protective rate obviously is superior to negative control group, and the egg capsule of discharge obviously reduces, and efflux time also reduces (seeing accompanying drawing 3).Humoral immunization testing result confirmation one is exempted from the back experimental group and is compared the variation of IgG titre with matched group not quite.Two exempt from, three exempt from back IgG titre and rise gradually, attack worm after the experimental group antibody titer to the highest, compare extremely significantly (P＜0.01) (seeing attached list 1) of difference with negative control group.Cellular level immune detection result shows immune group CD4
+, CD8
+Variable is apparently higher than negative control group, with all extremely remarkable (P＜0.01) (the seeing attached list 2) of negative control group difference.
Table 1 is respectively organized mice serum specific antibody IgG titre situation of change
Table 2 test group and control group mice spleen CD4
+And CD8
+The T lymphocyte quantity
Comprehensive above-mentioned experimental result, cryptosporidium parvum divalent protein vaccine pET28-CP12-CP21 of the present invention, higher to the Cryptosporidum parvum immune protective rate, can be used as Cryptosporidum parvum sick prevention and therapeutic vaccine.
Sequence table
CTGGATCCATGTCAGATGCATCAATA；
GGGGCGAATTCTATTTGTTCATTCATCTG
CCGAATTCGGTGGCGGTGGCTCGATGTCTAAAAGAGCAT
CTGGCTCTCGAGCTATTCATCTGTTTGAAC
Claims (3)
1. cryptosporidium parvum divalent protein vaccine; It is characterized in that: with prokaryotic expression carrier pET28a is carrier; CP12, two Cryptosporidum parvum protective antigen genes of CP21 are connected and are inserted into the MCS district of pET28a prokaryotic expression carrier, carry out prokaryotic expression, obtain vaccine.
2. the preparation technology of the said cryptosporidium parvum divalent protein vaccine of claim 1 may further comprise the steps:
Read frame design primer according to the opening of Cryptosporidum parvum protective antigen gene sequence and carry out PCR, the TA clone; CP12 and two protective antigen gene series connection of CP21 of gained are cloned into prokaryotic expression carrier pET28a carrier, carry out prokaryotic expression, obtain vaccine.
3. the preparation technology of the said cryptosporidium parvum divalent protein vaccine of claim 2 may further comprise the steps:
CP12, CP21 genes of interest are connected with the T carrier; Carry out the double digestion reaction with BamHI, EcoR1 and EcoR1, Xho1 respectively; Reclaim fragment and be connected with the prokaryotic expression carrier pET28a of Xho1 double digestion reaction through BamHI; Make up the pET28-CP12-CP21 recombinant vector, carry out prokaryotic expression, obtain vaccine.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880328B (en) * | 2010-06-02 | 2012-08-01 | 中国农业科学院上海兽医研究所 | Th multi-epitope gene and fusion protein of cryptosporidium parvum and application thereof |
| CN102276725B (en) * | 2010-06-13 | 2013-06-05 | 中国农业科学院上海兽医研究所 | Cryptosporidium CTL and Th mixed multi-epitope gene and fusion protein and application |
| CN102600479A (en) * | 2010-12-17 | 2012-07-25 | 吉林大学 | Cryptosporidium andersoni nucleic acid vaccine with cross protection and preparation method of vaccine |
| CN102221619A (en) * | 2011-06-07 | 2011-10-19 | 吉林大学 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection method and kit of cryptosporidium virus capsid protein antibody |
| CN110407944B (en) * | 2018-04-29 | 2023-04-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Cryptosporidium multi-epitope gene fragment cpmcef, fusion protein and application thereof |
| CN111537715B (en) * | 2020-07-09 | 2020-10-30 | 北京维德维康生物技术有限公司 | Cryptosporidium parvum antibody detection test strip and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| Longquan Yao et al..Cryptosporidium parvum: IdentiWcation of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library.《Experimental Parasitology》.2007,第115卷333–338. * |
| 何宏轩等.用微小隐孢子虫子孢子表面蛋白DNA疫苗免疫小鼠的实验研究.《中国兽医杂志》.2003,第39卷(第2期),3-5. * |
| 姚龙泉.微小隐孢子虫粘附相关蛋白基因的筛选、表达及免疫保护性研究.《中国博士学位论文全文数据库》.2006,全文. * |
| 陈玉兰.微小隐孢子虫表面粘附相关蛋白基因的筛选和免疫特性研究.《中国优秀硕士学位论文全文数据库》.2007,全文. * |
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