CN110420322A - The DC vaccine for the trichina ES co-induction agent induction for preventing, treating and diagnosing for pigs trichina disease - Google Patents

Abstract

The invention relates to a DC vaccine induced by a Trichinella ES compound inducer for the prevention, treatment and diagnosis of swine trichinosis, and belongs to the field of biotechnology. Including the preparation of Trichinella sp., adding 0.61 μg and 0.25 μg of camptothecin and hypericum respectively, the construction of porcine Trichinella DC vaccine, the isolation of porcine peripheral blood mononuclear cells, the acquisition of adherent cells, and the acquisition of DC vaccine. Advantages: The use of porcine peripheral blood mononuclear cells, the use of complete medium to induce DC maturation using the Trichinella sp. ES compound preparation, and the immunotherapy of Trichinella-infected meat-eating pigs. The Trichinella ES composite inducer is camptothecin and hypericin, which can enhance the induction effect of Trichinella exudates on DC cells. The invention can be used for preventing, treating and diagnosing pig trichinosis, and improving the quality of pork import and export. , to ensure food safety, reduce the economic losses caused by swine trichinosis to the pig industry, and avoid the negative impact of swine trichinosis on food safety and international pork trade.

Description

Trichinella ES compound inducer for swine trichinosis prevention, treatment and diagnosis DC vaccine

technical field

The invention belongs to the field of biotechnology, and particularly refers to a DC vaccine used for the prevention, treatment and diagnosis of swine trichinosis.

Background technique

Porcine trichinosis not only affects people's health, but also causes huge economic losses to the pig industry, and directly brings negative reputation in international trade.

With the development of international meat trade and tourism, as well as the improvement of the ecological environment such as afforestation and returning farmland to forests, coupled with the improvement of people's living standards, food diversification and excessive pursuit of original ecological products and other consumption factors stimulated by consumption factors in recent years Trichinellosis as a whole showed new characteristics: 1. The incidence rate in areas where raw meat was customary decreased, while the incidence in areas where cooked meat was eaten increased; 2. Local outbreaks were the main source of infection. The current control situation presents new challenges to the control of trichinosis. The use of albendazole as a swine feed additive has a good preventive effect on swine trichinosis, but the drug residues and side effects of albendazole in pork are still unclear, so the use of deworming drugs (such as Albendazole) is not recommended for the time being. benpyrazole) to deworming pigs, and the International Commission on Trichinella (ICT) also disapproves of this practice.

Antigen presenting cells (APCs) uptake, process and present antigens are the key links in initiating adaptive immune responses. Dendritic cells (DCs) are now recognized as the most powerful professional antigens in vivo. Presenting cells, on the one hand, can initiate and regulate cellular immunity and T cell-dependent specific humoral immune responses through antigen presentation and secretion of cytokines, and on the other hand, they can also induce regulatory T cells or eliminate corresponding T cell clones. The role of immunosuppression or induction of immune tolerance, DC is currently considered to be a key link between innate immunity and adaptive immunity, and it also plays a crucial role in maintaining immune balance. In recent years, with the development of DC vaccine application research, DC vaccine has become one of the focuses of attention today. At present, the types of DC vaccines mainly include antigen-sensitized DC vaccines, antigen-polypeptide-sensitized DC vaccines, and genetically modified DC vaccines. Compared with the first two types of DC vaccines, genetically modified DC vaccines have many advantages, which has attracted more and more scholars' attention. Although more than a dozen DC vaccines are in the clinical phase III research stage in recent years, which greatly stimulates the further development of DC vaccines, it also shows that DC vaccines have broad application prospects. At present, there is no porcine DC vaccine induced by the Trichinella ES co-formulation.

SUMMARY OF THE INVENTION

The invention provides a DC vaccine induced by a Trichinella ES compound inducer for the prevention, treatment and diagnosis of swine trichinosis.

The technical scheme adopted by the present invention is: comprising the following steps:

(1) Preparation of Trichinella ES composite inducer

(1) Trichinella ES preparation

Oral infection with 300 Trichinella worms and 10 Wistar rats were sacrificed by cervical dislocation, the small intestine was removed, the adipose tissue and mesentery were removed, the small intestine was longitudinally dissected, and the intestinal contents were washed with running tap water, and 300 grams of small intestine was added with 200 U/ml at 37°C. Wash the small intestine in 2000ml of normal saline containing penicillin and 200U/ml streptomycin sulfate, collect and count the adult Trichinella worms by artificial digestion; pour 50ml of the liquid containing 50,000 adult Trichinella worms into a clean glass dish with a diameter of 15cm, Let stand for 15min, aspirate the supernatant liquid, wash the collected adult Trichinella worms and transfer them to a 15ml centrifuge tube, wash twice with RPMI-1640 medium containing double antibody at 1000rpm/min, 2min centrifugation for 2 times, and then precipitate the washed pellets. The adult Trichinella worms at the bottom of the centrifuge tube were transferred to a 25cm ³ cell culture flask, 15ml medium, 37°C, 5% CO ₂ incubator for 12h, centrifuged after 12h, 1000rpm/min, 2min, and the centrifugation supernatant was collected; Repeated centrifugation of the supernatant overnight, 9°C, 6000rpm/min, 30min, concentrated until the ES liquid became 8-10ml colorless liquid, that is, Trichinella worm ES, transferred the colorless liquid to a 1.5mL centrifuge tube, sealed, -20 ℃ preservation;

(2) Preparation of Trichinella ES composite inducer

0.61μg of camptothecin and hypericum were added to each ml of ES respectively, and 0.25μg was used for later use;

The preparation of camptothecin and hypericin, the medicine is prepared to 200mg/mL according to the storage concentration, and diluted to 0.1ug/ml when used;

(2) Construction of porcine Trichinella DC vaccine

(1) Isolation of porcine peripheral blood mononuclear cells:

Take 10ml of pig anterior external venous blood under sterile conditions, aspirate the upper serum for later use, add an equal volume of PBS to dilute the blood, and use a pipette to slowly add 14ml of diluted peripheral blood to a centrifuge tube containing 7ml of NycoPrepTM 1.077A lymphocyte separation solution along the tube wall. medium, centrifuge at 1800rpm for 20min, gently suck out the interface cells in the centrifuge tube to separate the mononuclear cells with a pipette, transfer it to another sterile centrifuge tube, gently pipette PBS to make it fully diluted, centrifuge at 1500rpm for 8min, wash twice, to collect monocytes;

(2) Acquisition of adherent cells

The mononuclear cells obtained were suspended in RPM11640 solution containing 10% fetal bovine serum, pipetted evenly, 10ul of cell suspension was added with 90ul of trypan blue, mixed well, and then added to the counting plate for live cell counting. Hope blue staining demonstrated cell viability >95%,

Calculation formula: number of cells/L=total number of live cells in 4 large cells/4×104×dilution factor

Adjust the concentration of monocytes to 2×10 ⁶ cells/ml, add 2 ml per well to a 6-well cell culture plate, incubate at 37°C with 5% CO ₂ , wash off non-adherent cells after 90 min, and add FCS-free cells. The RPMI1640 was incubated for 30 min, and the non-adherent cells were washed away again after 30 min, and the remaining cells were adherent monocytes;

(3) Obtaining DC vaccine

Add 2ml of complete medium (recombinant colony stimulating factor (rmGSF) 160ng/ml + rmIL-4 50ng/ml + 10% fetal bovine serum) to each well, put the culture plate in a sterile plastic bag, wrap the bag mouth, and place the cells at 5 Cultivated at 37°C with % CO ₂ , washed the culture wells with a pipette after 7 days, sucked the cell suspension into a centrifuge tube, centrifuged at 1500 rpm for 8 min, and collected the centrifugation pellet; the centrifuged pellet obtained by culture in complete medium, the medium was changed on the third day , on the sixth day, add 5ml of Trichinella ES compound inducer, on the seventh day, add TNF-α100u/ml to induce maturation, pipetting and mixing, all the collected suspension cells are porcine Trichinella spp DC, and the suspension cells are counted to make the suspension cells The concentration is 2×10 ⁶ /ml, that is, the pig Trichinella DC vaccine; the dosage of pig per 100Kg body weight is 1 ml.

According to the advantages of the invention, the porcine peripheral blood mononuclear cells are used, and the complete medium is used to induce DC maturation by using the Trichinella sp. ES compound preparation to perform immunotherapy on the meat-eating pigs infected with Trichinella spiralis. The trichinella ES compound inducer used is derived from icariin, podophyllotoxin, kaempferol, chlorogenic acid, oleanolic acid, emodin, quercetin, forsythiaside, camptothecin, chrysophanol , indirubin, indigo, hypericin, proanthocyanidins, nepeta oil and other 15 kinds of active ingredients of traditional Chinese medicine were screened and obtained, and it was confirmed that camptothecin and hypericin can enhance the secretion of Trichinella spiralis (ES) on DC cells. Induction, the present invention can be used to prevent, treat and diagnose swine trichinosis, improve the quality of pork import and export, ensure food safety, reduce the economic loss caused by swine trichinosis to the pig industry, and avoid the effect of swine trichinosis on food safety. and the negative impact of pork international trade.

Description of drawings

Figure 1 is a graph of DCs induced by ES complex inducers and DCs without ES complex inducers;

Fig. 2 is a flow cytometric identification diagram of mature DC induced by ES compound inducer;

Figure 3 is a diagram showing the regulation of porcine blood cytokines by DC induced by Trichinella ES composite inducer;

Fig. 4 is the hypericin concentration screening diagram of the present invention;

Fig. 5 is the screening chart of the camptothecin concentration of the present invention.

Detailed ways

Include the following steps:

(1) Preparation of Trichinella ES composite inducer

(1) Trichinella ES preparation

Oral infection with 300 Trichinella worms and 10 Wistar rats were sacrificed by cervical dislocation, the small intestine was removed, the adipose tissue and mesentery were removed, the small intestine was longitudinally dissected, and the intestinal contents were washed with running tap water, and 300 grams of small intestine was added with 200 U/ml at 37°C. Wash the small intestine in 2000ml of normal saline containing penicillin and 200U/ml streptomycin sulfate, collect and count the adult Trichinella worms by artificial digestion; pour 50ml of the liquid containing 50,000 adult Trichinella worms into a clean glass dish with a diameter of 15cm, Let stand for 15min, aspirate the supernatant liquid, wash the collected adult Trichinella worms and transfer them to a 15ml centrifuge tube, wash twice with RPMI-1640 medium containing double antibody at 1000rpm/min, 2min centrifugation for 2 times, and then precipitate the washed pellets. The adult Trichinella worms at the bottom of the centrifuge tube were transferred to a 25cm ³ cell culture flask, 15ml medium, 37°C, 5% CO ₂ incubator for 12h, centrifuged after 12h, 1000rpm/min, 2min, and the centrifugation supernatant was collected; Repeated centrifugation of the supernatant overnight, 9°C, 6000rpm/min, 30min, concentrated until the ES liquid became 8-10ml colorless liquid, that is, Trichinella worm ES, transferred the colorless liquid to a 1.5mL centrifuge tube, sealed, -20 ℃ preservation;

(2) Preparation of Trichinella ES composite inducer

0.61μg of camptothecin and hypericum were added to each ml of ES respectively, and 0.25μg was used for later use;

The preparation of camptothecin and hypericin, the medicine is prepared to 200mg/mL according to the storage concentration, and diluted to 0.1ug/ml when used;

(2) Construction of porcine Trichinella DC vaccine

(1) Isolation of porcine peripheral blood mononuclear cells:

Take 10ml of pig anterior external venous blood under sterile conditions, aspirate the upper serum for later use, add an equal volume of PBS to dilute the blood, and use a pipette to slowly add 14ml of diluted peripheral blood to a centrifuge tube containing 7ml of NycoPrepTM 1.077A lymphocyte separation solution along the tube wall. medium, centrifuge at 1800rpm for 20min, gently suck out the interface cells in the centrifuge tube to separate the mononuclear cells with a pipette, transfer it to another sterile centrifuge tube, gently pipette PBS to make it fully diluted, centrifuge at 1500rpm for 8min, wash twice, to collect monocytes;

(2) Acquisition of adherent cells

The mononuclear cells obtained were suspended in RPM11640 solution containing 10% fetal bovine serum, pipetted evenly, 10ul of cell suspension was added with 90ul of trypan blue, mixed well, and then added to the counting plate for live cell counting. Hope blue staining demonstrated cell viability >95%,

Calculation formula: number of cells/L=total number of live cells in 4 large cells/4×104×dilution factor

Adjust the concentration of monocytes to 2×10 ⁶ cells/ml, add 2 ml per well to a 6-well cell culture plate, incubate at 37°C with 5% CO ₂ , wash off non-adherent cells after 90 min, and add FCS-free cells. The RPMI1640 was incubated for 30 min, and the non-adherent cells were washed away again after 30 min, and the remaining cells were adherent monocytes;

(3) Obtaining DC vaccine

Add 2ml of complete medium (recombinant colony stimulating factor (rmGSF) 160ng/ml + rmIL-4 50ng/ml + 10% fetal bovine serum) to each well, put the culture plate into a sterile plastic bag, wrap the bag mouth, and place the cells in 5 Cultivated at 37°C with % CO ₂ , washed the culture wells with a pipette after 7 days, sucked the cell suspension into a centrifuge tube, centrifuged at 1500 rpm for 8 min, and collected the centrifugation pellet; the centrifuged pellet obtained by culture in complete medium, the medium was changed on the third day , on the sixth day, add 5ml of Trichinella ES compound inducer, on the seventh day, add 100u/ml of TNF-α to induce maturation, mix by pipetting, collect all suspended cells, count the suspended cells, and make the suspended cell concentration 2×10 ⁶ per 100Kg body weight of pigs is 1ml.

Experimental example 1. Morphological identification

1. Identification by phase contrast inverted microscope: cell growth state and morphological changes were observed every day, and cell photos were taken on the 7th day.

2. Scanning electric field identification: centrifuge the collected suspension cells, remove 2/3 of the supernatant, and resuspend the cells; aspirate the cell suspension, carefully drop it on the glass slide covered with the film, and place it in a petri dish to stand still 30min, then add 2.5% glutaraldehyde to the petri dish, immerse the slides in it, fix for 30min, rinse with 0.1M buffer, fix with 1% osmic acid for 30min, rinse with buffer, start gradient dehydration with 30% alcohol, Hexamyl acetate was transitioned for 5 min, and the critical point was dry. The glass slide was taken out and pasted on the specimen table with conductive glue, and ion sputtered coating; scanning electric field observation and photographing.

Transmission electric field identification: cell pellets were obtained by centrifugation, fixed with 4% glutaraldehyde for 2 h (prefixed), and rinsed with 0.1 M phosphate buffer. After fixation in 1% osmic acid for 2h, rinsed with 0.1M phosphate buffer, gradient dehydration with 30% ethanol; acetone, resin replacement and infiltration, embedding and polymerization, trimming section, uranyl acetate, lead citrate staining; transmission electric observation Photograph. As shown in Figure 1 (B and D), the mature DCs induced by ES composite inducers have long and numerous dendrites on the surface, with a large number of folds, showing dendrites with different thickness, size and length, some of which are relatively straight. Some are more curved, covering the entire cell, and the cell surface is not smooth and uneven. Consistent with the morphological characteristics of typical mature DCs.

Figure 1 (A, C) shows immature DC cells without inducers.

3. Flow cytometric identification of DC vaccine

The expression of CD86 in mature DC and immature DC cells was detected by flow cytometry (Fig. 2E,B), CD11c is a characteristic surface molecule of myeloid-derived DC, and CD11c+ indicates that the extracted cells are myeloid-derived, not the source in the lymphatic system. Both immature and mature DCs highly expressed CD11c (Fig. 2A,D), whereas mature DCs expressed higher amounts of MHC-II (Fig. 2F,C) and CD86 than immature counterparts.

Experimental example 2. Immunotherapy of swine trichinosis

1. ES complex inducer induces DC vaccine deworming rate

40 healthy pigs were infected with 2000 Trichinella worms (Trichinella International No. ISS534)/head, respectively, and 10 pigs were infected with Trichinella by induction of DC vaccine with normal saline, DC, ES and ES complex inducers respectively; every 100Kg body weight of pigs The usage amount is 1ml;

Immunization strategy: After infection, all treated pigs were immunized with DC vaccine at a single dose on day 0 and 2, double-dose immunization on day 7, and double-dose immunization on day 13 and 17. treatment of pigs;

After 18 days, the pigs of each group were slaughtered, and the muscle and intestinal Trichinella worms of each group of pigs were collected and counted by digestion method, and the insect reduction rate was calculated according to the following formula. The results are shown in Table 1.

Reduction rate = (average number of trichinella in saline treatment group - average number of trichinella in treatment group)/average number of trichinella in saline treatment group

Table 1 Deworming rate of DC vaccine induced by ES complex inducer

group normal saline DC ES ES complex inducers induce DCs Insect reduction rate (%) 28.53 35.71 44.82 48.41

2. The regulation of porcine blood cytokines by DC induced by Trichinella ES complex inducer

40 healthy pigs were infected with 2000 Trichinella worms (Trichinella International No. ISS534)/head, respectively, and 10 pigs were infected with Trichinella by induction of DC vaccine with normal saline, DC, ES and ES complex inducers respectively, and each pig was 100Kg body weight. The amount used was 1 ml; blood was collected from the vena cava before 3, 6, 9, 12, 15, 18, 21, 24, and 27 days respectively, and the changes of cytokines in serum were detected by ELISA according to the instructions of the eBioscience kit.

Immune effect evaluation:

The changes of various cytokines are shown in Figure 4. The expression levels of IL-6 (Fig. 3B), IL-4 (Fig. 3C) and IL-10 (Fig. 3D) representing the immune response of Trichinella spiralis were down-regulated, and IFN-γ (Fig. 3A) was up-regulated.

Experimental example 3. Screening of hypericin and camptothecin concentrations

Hypericin and camptothecin were treated with porcine fetal fibroblasts (G418) in six concentration groups of 8 μg/mL, 1.6 μg/mL, 0.32 μg/mL, 0.064 μg/mL, and each concentration was set for 24 hours. , 48h, 72h three time groups, and set up solvent control wells and blank wells, each group set up 3 parallel wells. The drug concentration with 5% toxicity to cells (ie, the drug concentration when the cell viability rate is 95%) was taken as the substantially non-toxic concentration of the drug to cells, and the subsequent experiments were carried out with this concentration at 24 h. Cell survival rate=(OD value experimental group-OD value control group)/(OD value control group-OD value blank group)×100%. The results are shown in Figures 4 and 5.

Claims (4)

1. a DC vaccine induced by the Trichinella ES composite inducer for porcine trichinosis prevention, treatment and diagnosis, is characterized in that: comprise the following steps: (1) Preparation of Trichinella ES composite inducer (1) Trichinella ES preparation Oral infection with 300 Trichinella worms and 10 Wistar rats were sacrificed by cervical dislocation, the small intestine was removed, the adipose tissue and mesentery were removed, the small intestine was longitudinally dissected, and the intestinal contents were washed with running tap water, and 300 grams of small intestine was added with 200 U/ml at 37°C. Wash the small intestine in 2000ml of normal saline containing penicillin and 200 U/ml streptomycin sulfate, collect and count the adult Trichinella spiralis by artificial digestion; pour 50ml of the liquid containing 50,000 adult Trichinella spiralis into a clean glass dish with a diameter of 15cm , let stand for 15min, aspirate the supernatant liquid, wash the collected adult Trichinella worms and transfer them to a 15ml centrifuge tube, use RPMI-1640 culture medium containing double antibody at 1000rpm/min, 2min centrifugation and wash 2 times. The adult Trichinella worms deposited at the bottom of the centrifuge tube were transferred to a 25cm ³ cell culture flask, 15ml medium, 37°C, 5% _CO2 incubator for 12h, centrifuged after 12h, 1000rpm/min, 2min, and the centrifugation supernatant was collected ; Repeated centrifugation of the supernatant overnight, 9°C, 6000rpm/min, 30min, concentrated until the ES liquid became 8-10ml colorless liquid, that is, Trichinella sp. Transfer the colorless liquid to a 1.5mL centrifuge tube, seal, - Store at 20°C; (2) Preparation of Trichinella ES composite inducer 0.61μg of camptothecin and hypericum were added to each ml of ES respectively, and 0.25μg was used for later use; (2) Construction of porcine Trichinella DC vaccine (1) Isolation of porcine peripheral blood mononuclear cells: Take 10ml of pig anterior external venous blood under sterile conditions, aspirate the upper serum for later use, add an equal volume of PBS to dilute the blood, and use a pipette to slowly add 14ml of diluted peripheral blood to a centrifuge tube containing 7ml of NycoPrepTM 1.077A lymphocyte separation solution along the tube wall. medium, centrifuge at 1800rpm for 20min, gently suck out the interface cells in the centrifuge tube to separate the mononuclear cells with a pipette, transfer it to another sterile centrifuge tube, gently pipette PBS to make it fully diluted, centrifuge at 1500rpm for 8min, wash twice, to collect monocytes; (2) Acquisition of adherent cells The mononuclear cells obtained were suspended in RPM11640 solution containing 10% fetal bovine serum, pipetted evenly, 10ul of cell suspension was added with 90ul of trypan blue, mixed well, and then added to the counting plate for live cell counting. Hope blue staining demonstrated cell viability >95%, Calculation formula: number of cells/L=total number of live cells in 4 large cells/4×104×dilution factor Adjust the concentration of monocytes to 2×10 ⁶ cells/ml, add 2 ml per well to a 6-well cell culture plate, incubate at 37°C with 5% CO ₂ , wash off non-adherent cells after 90 min, and add FCS-free cells. The RPMI1640 was incubated for 30 min, and the non-adherent cells were washed away again after 30 min, and the remaining cells were adherent monocytes; (3) Obtaining DC vaccine Add 2ml of complete medium to each well, put the culture plate into a sterile plastic bag, wrap the bag mouth, and culture the cells at 5% CO ₂ at 37°C. After 7 days, the culture wells are thoroughly washed with a pipette, and the cell suspension is aspirated into a centrifuge tube. medium, centrifuge at 1500 rpm for 8 min, and collect the centrifuged precipitate; the centrifuged precipitate obtained by culture in complete medium, change the medium on the 3rd day, add 5ml of Trichinella ES compound inducer on the sixth day, and add TNF-α 100u/ml on the seventh day to induce maturation. , pipetting and mixing, all the collected suspension cells are Trichinella porcine DC, and the suspended cells are counted to make the suspension cell concentration 2×10 ⁶ cells/ml, which is the Trichinella porcine DC vaccine. 2. a kind of DC vaccine induced by the Trichinella ES composite inducer for swine trichinosis prevention, treatment and diagnosis according to claim 1, is characterized in that: step (1), Trichinella ES composite inducer are prepared (2) In the preparation of the Trichinella ES composite inducer, camptothecin and hypericin were prepared, and the drug was prepared to 200 mg/mL according to the storage concentration, and diluted to 0.1 ug/ml when used. 3. a kind of DC vaccine induced by the Trichinella ES composite inducer for swine trichinosis prevention, treatment and diagnosis according to claim 1, it is characterized in that: step (2), swine trichinella DC vaccine constructs (3) In obtaining the DC vaccine, the complete medium added to each well is: recombinant colony stimulating factor (rmGSF) 160ng/ml+rmIL-4 50ng/ml+10% fetal bovine serum. 4. a kind of DC vaccine induced by the Trichinella ES compound inducer for the prevention, treatment and diagnosis of swine trichinosis according to claim 1, is characterized in that: the usage amount per 100Kg of pig body weight is 1ml.