CN114028558A - A kind of preparation method using bacterial lysate as inactivated vaccine adjuvant - Google Patents
A kind of preparation method using bacterial lysate as inactivated vaccine adjuvant Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
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- B04B5/00—Other centrifuges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
- A61K2039/55594—Adjuvants of undefined constitution from bacteria
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Abstract
本发明的一种以细菌裂解物作为灭活疫苗佐剂的制备方法，包括如下步骤：步骤1，将伪结核棒状杆菌和马红球菌分别置LB培养基37℃培养后，通过离心机收获菌体；步骤2，将各菌体用灭菌生理盐水稀释到8.0×109cfu/mL，对菌液采用高压匀质仪破碎；步骤3，经无菌检验后，将伪结核棒状杆菌和马红球菌的裂解产物按照1:1比例与甲醛灭活的两种菌体混合，得到添加有佐剂的灭活疫苗。其具有安全、高效、免疫增强能力强等特点，并且成本低，适合用于多种细菌灭活疫苗。
A preparation method of using bacterial lysate as an inactivated vaccine adjuvant of the present invention includes the following steps: Step 1, after culturing Corynebacterium pseudotuberculosis and Rhodococcus equi respectively in LB medium at 37°C, harvest the bacteria through a centrifuge Step 2, dilute each bacterial cell with sterile physiological saline to 8.0×10 9 cfu/mL, and use a high-pressure homogenizer to crush the bacterial liquid; Step 3, after sterility test, remove Corynebacterium pseudotuberculosis and equine The lysate of Rhodococcus was mixed with two forms of bacteria inactivated by formaldehyde in a ratio of 1:1 to obtain an inactivated vaccine with adjuvant added. It has the characteristics of safety, high efficiency, strong immune-enhancing ability, and low cost, and is suitable for use in a variety of bacterial inactivated vaccines.
Description
Technical Field
The invention belongs to the field of animal husbandry and veterinary medicine, and particularly relates to a preparation method of an inactivated vaccine adjuvant by using a bacterial lysate.
Background
Rhodococcus, Nocardiaceae, Rhodococcus, order Rhodococcus equi. The bacterium is a gram-positive facultative intracellular parasitic bacterium, the bacterial colony is pink or yellow, rod-shaped and spherical, acid-resistant, free of spores and mostly free of flagella, but some bacterial strains have pili or appendages. The cell wall of rhodococcus equi is hydrophobic and surrounded by a polysaccharide capsule, the major components of which are lipids rich in mycolic acid (mycolicids) and lipopolysaccharides, wherein the lipopolysaccharides are Trehalose Dimycolate (TDM) and Lipoarabinomannan (LAM).
Rhodococcus equi is capable of causing host macrophages to undergo translocation of NF-. kappa.B and activate the production of a variety of pro-inflammatory mediators, such as tumor necrosis factor (TNF-. alpha.), interleukin-12 (IL-12) and Nitric Oxide (NO), via Toll-like receptor 2(TLR 2). The low-dose virulent strain of the rhodococcus equi can cause the colt to generate stronger Th1 immune response to the rhodococcus equi; in adult horses, Rhodococcus equi causes a Th1 response characteristic of IFN- γ production, and IL-12 production by activated macrophages and dendritic cells also stimulates the differentiation of Th0 cells into Th1 cells. The Rhodococcus equi has the capability of activating the immune system of the organism and has the potential to be used as a vaccine adjuvant.
Corynebacterium pseudotuberculosis (Corynebacterium pseudotuberculosis) is a gram-positive facultative intracellular pathogen belonging to the genus Actinomyces. The bacterium is facultative anaerobic, contains mycolic acid in its cell, is in polymorphous form, does not form spore or spore, does not move, and has pilus. The bacterium is characterized in that the bacterium has a cell wall tissue, wherein the cell wall mainly comprises peptidoglycan, arabinouronic acid and mycorrhizal acid; (2) the G + C content of the genome is high (47-74%).
The pseudotuberculosis corynebacterium sp causes a significant increase in the expression levels of IL-1 β and TNF- α. IL-1 β is a very important proinflammatory cytokine, playing a central role in local and systemic infections and injuries; TNF-alpha plays an important role in host resistance to infection by Corynebacterium pseudotuberculosis, is an important inflammatory mediator of innate immune response, and has the effects of inducing cytokine production, activating expression of adhesion molecules and growth stimulation, and inducing inflammatory response to resist infection and immunoregulation.
The PLD gene in Corynebacterium pseudotuberculosis can also activate complement and promote differentiation of neutrophils. In view of the activation effect of the corynebacterium pseudotuberculosis on the immune system of the organism, the corynebacterium pseudotuberculosis is used as an adjuvant to improve the immune effect of the inactivated vaccine, and is theoretically feasible.
The currently used vaccine adjuvants, such as white oil adjuvant, usually cause local stimulation reaction, have the disadvantages of weak induced immune response and poor immune enhancement effect, and limit the use of the vaccine adjuvants, so that a new generation of efficient, low-toxicity and cheap vaccine adjuvants needs to be developed.
Disclosure of Invention
Therefore, the invention provides a preparation method of taking bacterial lysate as an inactivated vaccine adjuvant, which comprises the following steps:
and 3, after the aseptic examination, mixing the lysate of the corynebacterium pseudotuberculosis and the rhodococcus equi with two bacteria inactivated by formaldehyde according to the proportion of 1:1 to obtain the inactivated vaccine added with the adjuvant.
Preferably, in step 2, the bacterial liquid is broken three times by using a high-pressure homogenizer.
Preferably, in the step 3, the inactivated vaccine is subjected to physical property inspection, aseptic inspection and formaldehyde residue inspection, and meanwhile, the inactivated vaccine protection efficiency test is performed to detect the safety and the protection efficacy of the vaccine; the vaccine is finally preserved at 2-8 ℃ through physical property inspection, sterility inspection, standard exceeding inspection, overdose immunity safety inspection and efficacy inspection.
Preferably, the centrifuge comprises: a box body, a motor, a first rotating shaft, a first gear, a second rotating shaft, a second disc, a second gear and a supporting plate,
a motor is arranged at the bottom of the inner wall of the box body, an output shaft of the motor is upwards connected with one end of a first rotating shaft in the vertical direction, the other end of the first rotating shaft is coaxially connected with a first disc, a first gear is arranged on the first rotating shaft, a second rotating shaft in the vertical direction is arranged at the bottom of the inner wall of the box body, one end of the second rotating shaft is rotatably connected with the inner wall of the box body, a second disc is arranged at the other end of the second rotating shaft, a second gear is arranged on the second rotating shaft, the first gear and the second gear have the same diameter and are meshed with each other, a horizontal supporting plate is arranged in the box body, the first rotating shaft and the second rotating shaft both penetrate through the supporting plate and are rotatably connected with the supporting plate, and the supporting plate is positioned between the first gear and the first disc;
the diameter of first disc and second disc equals, be provided with first eccentric post on the first disc, be provided with the eccentric post of second on the second disc, first eccentric post and the eccentric post symmetry of second set up, first eccentric capital end is rotated and is connected with first slider, the eccentric post upper end of second is provided with the connecting rod, connecting rod one end with the eccentric post upper end of second is rotated and is connected, the connecting rod other end passes first slider, and with first slider sliding connection, first slider top is provided with the direction guide way of horizontal direction, spliced pole one end with the connecting rod is connected, and the spliced pole other end passes the direction guide way is connected with the workstation, the workstation top is provided with a plurality of standing grooves.
Preferably, a cover is arranged at the top end of the workbench and is in threaded connection with the workbench.
Preferably, one end of the connecting rod, which is far away from the second eccentric column, is provided with a limiting block.
Preferably, a box door is arranged at the top end of the box body, and one end of the box door is hinged with the top end of the box body.
Preferably, the side wall of the box body is provided with a plurality of heat dissipation holes.
Preferably, the high pressure homogenizer comprises: a cylinder body, a push plate, a fixed pipe, a first connecting rod, an opening, a sliding pipe, a second connecting rod and a third connecting rod,
an electric push rod is arranged at the top of the inner wall of the barrel, the output end of the electric push rod is downwards connected with a push plate, a fixed pipe in the vertical direction is arranged below the push plate, the outer wall of the fixed pipe is connected with the inner wall of the barrel through a first connecting rod, openings in the vertical direction are respectively arranged on the left side and the right side of the outer wall of the fixed pipe, a slide pipe is arranged in the fixed pipe and can reciprocate up and down in the fixed pipe, second connecting rods in the horizontal direction are arranged at the two ends of the slide pipe, one end of each second connecting rod is connected with the outer wall of the slide pipe, the other end of each second connecting rod penetrates through the opening to be vertically connected with one end of a third connecting rod, and the other end of each third connecting rod is connected with the bottom end of the push plate;
a rotating pipe is arranged in the sliding pipe, a spiral sliding groove is formed in the outer wall of the rotating pipe, a sliding column is arranged on the inner wall of the rotating pipe, the sliding column can slide in the spiral sliding groove, the bottom end of the rotating pipe extends out of the sliding pipe and is connected with a rotating plate, a first annular sliding groove and a second annular sliding groove are formed in the inner wall of the lower end of the fixed pipe at intervals, the first annular sliding groove and the second annular sliding groove are communicated through a vertical groove, second sliding blocks are arranged on the left side and the right side of the rotating plate respectively, the second sliding blocks can slide in the vertical groove in a reciprocating mode up and down mode and can also rotate in the first annular sliding groove and the second annular sliding groove, a horizontal partition plate is arranged below the fixed pipe and is connected with the inner wall of the barrel body, a shaft hole is formed in the partition plate, and the bottom end of the rotating plate is connected with one end of a third rotating shaft, the other end of the third rotating shaft penetrates through the shaft hole to be connected with the cutting knife, and a liquid inlet pipe and a liquid outlet pipe are respectively arranged on the outer wall of the lower end of the barrel.
Preferably, the liquid inlet pipe and the liquid outlet pipe are both provided with high-pressure valves.
The technical scheme of the invention has the following advantages: the inactivated vaccine added with the adjuvant has biological safety, and the vaccine strain is selected to be physically crushed under high pressure, so that the inactivated vaccine has the function of improving the immune effect of the vaccine. The vaccine adjuvant has the characteristics of simple preparation method, low cost and good application prospect.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and drawings.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1A is a photograph under an optical microscope of Rhodococcus equi, FIG. 1B is a photograph under an electron microscope of Rhodococcus equi, FIG. 1C is a photograph under an optical microscope of Corynebacterium pseudotuberculosis, and FIG. 1D is a photograph under an electron microscope of Corynebacterium pseudotuberculosis;
FIG. 2 shows the antibody changes of mouse immune Pasteurella;
FIG. 3 shows the HI antibody changes of newcastle disease inactivated vaccine;
FIG. 4 is a schematic view of the centrifuge of the present invention;
FIG. 5 is a schematic view of the connection of the first slider and the connecting rod according to the present invention;
FIG. 6 is a schematic view of the structure of the high pressure homogenizer of the present invention;
FIG. 7 is a schematic sectional view of the fixing tube of the present invention;
FIG. 8 is a schematic structural view of a rotating plate and a second sliding block according to the present invention;
wherein, 1-box, 2-motor, 3-first rotating shaft, 4-first gear, 5-second rotating shaft, 6-second disk, 7-second gear, 8-supporting plate, 9-first eccentric column, 10-second eccentric column, 11-first slide block, 12-connecting rod, 13-guiding opening, 14-connecting rod, 15-workbench, 16-placing groove, 17-first disk, 18-cover, 19-limiting block, 20-box door, 21-radiating hole, 22-barrel, 23-push plate, 24-fixing tube, 25-first connecting rod, 26-opening, 27-sliding tube, 28-second connecting rod, 29-third connecting rod, 30-rotating tube, 31-spiral sliding groove, 32-sliding column, 33-rotating plate, 34-first annular chute, 35-second annular chute, 36-vertical chute, 37-second sliding block, 38-partition plate, 39-shaft hole, 40-third rotating shaft, 41-cutting knife, 42-liquid inlet pipe, 43-liquid outlet pipe and 44-high pressure valve.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly or indirectly connected to the other element.
It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, refer to an orientation or positional relationship indicated in the drawings that is solely for the purpose of facilitating the description and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and is therefore not to be construed as limiting the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Example one
Preparation of the adjuvant:
respectively inoculating corynebacterium pseudotuberculosis and rhodococcus equi to an LB solid culture medium at 37 ℃ for 14h, selecting a single colony, inoculating the single colony to 3mL of an LB liquid culture medium at 37 ℃ in a shaking table (220rpm) for 12h, inoculating 600mL of the LB liquid culture medium according to the proportion of 3 per thousand, culturing at 37 ℃ for 12h-16h, measuring OD600 to reach about 1.0, centrifugally collecting thalli, diluting with sterilized normal saline to ensure that the concentration of the thalli reaches 8.0 multiplied by 109cfu/mL, respectively and directly passing through a high-pressure homogenizer, crushing the thalli, or continuously crushing three times through the high-pressure homogenizer after 1:1 mixing to obtain the corynebacterium pseudotuberculosis, the rhodococcus equi and a mixed vaccine adjuvant, mixing the corynebacterium pseudotuberculosis, the rhodococcus equi and the inactivated pasteurella added with inactivated pasteurella 1:1, and obtaining the inactivated pasteurella added with the corynebacterium pseudotuberculosis, the rhodococcus equi and the mixed vaccine. The morphology of Corynebacterium pseudotuberculosis and Rhodococcus equi is shown in FIG. 1.
Sterile test of the immunologic adjuvant: and inoculating 200 mu L of the crushed thallus into 5mL of liquid culture medium, inoculating each sample into 3 test tubes, observing for 5 days, and performing inactivation safety inspection, wherein the inoculated liquid culture medium is still clear and has no turbidity after 5 days, and the safety inspection is qualified.
Example two
Evaluation of the safety of lysates of Corynebacterium pseudotuberculosis and Rhodococcus equi:
three batches of adjuvant prepared in example one were injected into 3 healthy mice in a single dose, 5-fold dose and 10-fold dose, and inoculated into the medial hind limb muscle. Observing for 14d, the inoculated mice are mild and have normal appetite, and the inoculated parts have no swelling and inflammation and have no local and systemic reaction; and (3) killing the mice after 14d, cutting the skin of the injection part, observing the local pathological changes of the injection, ensuring that the subcutaneous tissue of the inoculation part of the mice has no abnormal change, the muscle tissue of the intramuscular inoculation part on the inner side of hind limbs is normal, and the injection part has no inflammatory reaction. Thus indicating that the composition has better safety to mice.
Example three
The improvement effect of the corynebacterium pseudotuberculosis lysate and rhodococcus equi lysate on the mice immunized by the pasteurella inactivated vaccine:
mixing the corynebacterium pseudotuberculosis lysate, rhodococcus rhodochrous lysate and lysate of the two mixtures and 20% of alumina gel adjuvant with the pasteurella inactivated vaccine according to a ratio of 1:9 respectively, immunizing 5 and 1 ml/mouse respectively in 4-6 weeks old Kunming mice, continuously detecting the level of the antibody for 8 weeks by using an indirect ELISA method established in a laboratory after immunization, and drawing an antibody growth rule, wherein the antibody growth rule is shown in figure 2.
Example four
The improvement effect of the corynebacterium pseudotuberculosis and rhodococcus equi lysate newcastle disease virus inactivated vaccine immune chicks is as follows:
the corynebacterium pseudotuberculosis, rhodococcus equi lysate adjuvant and 20% alumina gel adjuvant are mixed with the inactivated vaccine of the newcastle disease virus according to the proportion of 1:9 respectively to immunize 20 chicks of 30 days old with 1 ml/chicken, the level of the antibody is continuously detected by an indirect ELISA method after immunization for 8 weeks, and the law of the antibody length reduction is drawn, as shown in figure 3.
The beneficial technical effects of the technical scheme are as follows: the inactivated vaccine added with the adjuvant has biological safety, and the vaccine strain is selected to be physically crushed under high pressure, so that the inactivated vaccine has the function of improving the immune effect of the vaccine. The vaccine adjuvant has the characteristics of simple preparation method, low cost and good application prospect.
In one embodiment, as shown in fig. 4-5, the centrifuge comprises: a box body 1, a motor 2, a first rotating shaft 3, a first gear 4, a second rotating shaft 5, a second disc 6, a second gear 7 and a supporting plate 8,
the bottom of the inner wall of the box body 1 is provided with a motor 2, an output shaft of the motor 2 is upwards connected with one end of a first rotating shaft 3 in the vertical direction, the other end of the first rotating shaft 3 is coaxially connected with a first disc 17, a first gear 4 is arranged on the first rotating shaft 3, a second rotating shaft 5 in the vertical direction is arranged at the bottom of the inner wall of the box body 1, one end of the second rotating shaft 5 is rotationally connected with the inner wall of the box body 1, the other end of the second rotating shaft 5 is provided with a second disc 6, the second rotating shaft 5 is provided with a second gear 7, the first gear 4 and the second gear 7 have the same diameter and are meshed with each other, a horizontal supporting plate 8 is arranged in the box body 1, the first rotating shaft 3 and the second rotating shaft 5 both penetrate through the supporting plate 8, and is rotatably connected with the supporting plate 8, the supporting plate 8 is positioned between the first gear 4 and the first disk 17;
the diameter of first disc 17 and second disc 6 equals, be provided with first eccentric post 9 on the first disc 17, be provided with second eccentric post 10 on the second disc 6, first eccentric post 9 and the eccentric 10 symmetry setting of second, 9 tops of first eccentric post are rotated and are connected with first slider 11, the eccentric 10 upper ends of second are provided with connecting rod 12, connecting rod 12 one end with the eccentric 10 upper ends of second rotate to be connected, the connecting rod 12 other end passes first slider 11, and with first slider 11 sliding connection, first slider 11 top is provided with the direction mouth 13 of horizontal direction, connecting rod 14 one end with connecting rod 12 is connected, and the connecting rod 14 other end passes direction mouth 13 is connected with workstation 15, workstation 15 top is provided with a plurality of standing grooves 16.
The working principle and the beneficial technical effects of the technical scheme are as follows: the liquid culture medium of the cultured corynebacterium pseudotuberculosis and rhodococcus equi is filled into a test tube, the test tube is placed in a placing groove 16, a motor 2 is started to drive a first rotating shaft 3 and a first gear 4 to rotate, the first rotating shaft 3 and a second rotating shaft 5 rotate in the same rotating speed and opposite directions through the meshing of the first gear 4 and a second gear 7, correspondingly, a first disc 17 and a second disc 6 rotate in the same rotating speed and opposite directions, a first eccentric column 9 and a second eccentric column 10 are always positioned on the same horizontal line, the positions of the first rotating shaft and the second rotating shaft are different and close to or far away from each other, a connecting rod 12 reciprocates in a first sliding block 11, and meanwhile, the first rotating shaft and the second eccentric column can rotate continuously, and thalli on the culture medium can be rapidly separated and collected through centrifugal force generated by left and right oscillation compound rotation.
In one embodiment, a cover 18 is arranged on the top end of the workbench 15, and the cover 18 is in threaded connection with the workbench 15 to ensure the workbench to be sealed.
In one embodiment, the end of the connecting rod 12 away from the second eccentric column 10 is provided with a stopper 19 to prevent the first sliding block 11 from sliding out of the connecting rod 12.
In one embodiment, the box body 1 is provided with a box door 20 at the top end, and one end of the box door 20 is hinged to the top end of the box body 1, so as to facilitate the opening or closing of the box door.
In one embodiment, the side wall of the box 1 is provided with a plurality of heat dissipation holes 21 for facilitating heat dissipation.
In one embodiment, as shown in fig. 6-8, the high pressure homogenizer comprises: a cylinder body 22, a push plate 23, a fixed pipe 24, a first connecting rod 25, an opening 26, a sliding pipe 27, a second connecting rod 28 and a third connecting rod 29,
an electric push rod is arranged at the top of the inner wall of the cylinder 22, the output end of the electric push rod is downwards connected with a push plate 23, a fixed pipe 24 in the vertical direction is arranged below the push plate 23, the outer wall of the fixed pipe 24 is connected with the inner wall of the cylinder 22 through a first connecting rod 25, openings 26 in the vertical direction are respectively arranged on the left side and the right side of the outer wall of the fixed pipe 24, a sliding pipe 27 is arranged in the fixed pipe 24, the sliding pipe 27 can reciprocate up and down in the fixed pipe 24, second connecting rods 28 in the horizontal direction are arranged at two ends of the sliding pipe 27, one end of each second connecting rod 28 is connected with the outer wall of the corresponding sliding pipe 27, the other end of each second connecting rod 28 penetrates through the opening 26 to be vertically connected with one end of a third connecting rod 29, and the other end of the third connecting rod 29 is connected with the bottom end of the push plate 23;
a rotating pipe 30 is arranged in the sliding pipe 27, a spiral sliding groove 31 is arranged on the outer wall of the rotating pipe 30, a sliding column 32 is arranged on the inner wall of the rotating pipe 30, the sliding column 32 can slide in the spiral sliding groove 31, the sliding pipe 27 extends out of the bottom end of the rotating pipe 30 to be connected with a rotating plate 33, a first annular sliding groove 34 and a second annular sliding groove 35 are respectively arranged on the inner wall of the lower end of the fixed pipe 24 at intervals, the first annular sliding groove 34 and the second annular sliding groove 35 are communicated through a vertical groove 36, second sliding blocks 37 are respectively arranged on the left side and the right side of the rotating plate 33, the second sliding blocks 37 can slide up and down in the vertical groove 36 in a reciprocating manner and also can rotate in the first annular sliding groove 34 and the second annular sliding groove 35, a horizontal partition plate 38 is arranged below the fixed pipe 24, and the partition plate 38 is connected with the inner wall of the cylinder 22, the partition plate 38 is provided with a shaft hole 39, the bottom end of the rotating plate 33 is connected with one end of a third rotating shaft 40, the other end of the third rotating shaft 40 penetrates through the shaft hole 39 to be connected with a cutting knife 41, and the outer wall of the lower end of the cylinder 22 is provided with a liquid inlet pipe 42 and a liquid outlet pipe 43 respectively. The liquid inlet pipe 42 and the liquid outlet pipe 43 are both provided with high pressure valves 44. The outer wall of the cylinder 22 is provided with a pressurizing port.
The working principle and the beneficial technical effects of the technical scheme are as follows: injecting sterilized bacterial liquid into the cylinder 22 through the liquid inlet pipe 42, closing the high-pressure valve 44 on the liquid inlet pipe, applying high pressure into the cylinder 22 through the pressurizing port, starting the electric push rod to drive the push plate 23 to reciprocate up and down, driving the second connecting rod 28, the third connecting rod 29 and the slide pipe 27 to reciprocate up and down, driving the slide pipe 27 to move upwards, driving the second slide block 37 of the rotating plate 33 to contact with the inner wall of the first annular chute 34, and along with the continuous upward movement of the slide pipe 27, the slide column 32 slides upwards in the spiral chute 31, so that the rotating pipe 30 rotates forwards until the slide column 32 slides to the uppermost end of the spiral chute 31; when the slide pipe 27 moves downwards, the rotating plate 33 and the second sliding block 37 are driven to move downwards, the rotating plate 33 and the second sliding block 37 move to the second annular sliding groove 35, the second sliding block 37 is in contact with the inner wall of the second annular sliding groove 35, the sliding column 32 slides downwards in the spiral sliding groove 31 along with the continuous downward movement of the slide pipe 27, so that the rotating pipe 30 rotates reversely, then moves upwards again, and circulates continuously, correspondingly, the rotating plate 33 can drive the third rotating shaft 40 and the cutting knife 41 to rotate in a forward and reverse alternating manner and can also reciprocate up and down, so that bacteria liquid in the lower end of the cylinder 22 can be cut and crushed fully, and all bacteria bodies can be mixed uniformly.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A preparation method of inactivated vaccine adjuvant using bacterial lysate is characterized by comprising the following steps:
step 1, respectively culturing corynebacterium pseudotuberculosis and rhodococcus equi in an LB (lysogeny broth) culture medium at 37 ℃, and then harvesting thalli through a centrifugal machine;
step 2, each cell was diluted to 8.0X 10 with sterilized physiological saline9cfu/mL, crushing the bacterial liquid by using a high-pressure homogenizer;
and 3, after the aseptic examination, mixing the lysate of the corynebacterium pseudotuberculosis and the rhodococcus equi with two bacteria inactivated by formaldehyde according to the proportion of 1:1 to obtain the inactivated vaccine added with the adjuvant.
2. The method for preparing an inactivated vaccine adjuvant from a bacterial lysate according to claim 1, wherein the bacterial solution is disrupted three times by a high pressure homogenizer in step 2.
3. The method for preparing the inactivated vaccine adjuvant by using the bacterial lysate according to claim 1, wherein in the step 3, the inactivated vaccine is subjected to physical property inspection, aseptic inspection and formaldehyde residue inspection, and meanwhile, an inactivated vaccine protection efficiency test is performed to detect the safety and the protection efficacy of the vaccine; the vaccine is finally preserved at 2-8 ℃ through physical property inspection, sterility inspection, standard exceeding inspection, overdose immunity safety inspection and efficacy inspection.
4. The method for preparing inactivated vaccine adjuvant from bacterial lysate according to claim 1, wherein the centrifuge comprises: a box body (1), a motor (2), a first rotating shaft (3), a first gear (4), a second rotating shaft (5), a second disc (6), a second gear (7) and a supporting plate (8),
a motor (2) is arranged at the bottom of the inner wall of the box body (1), an output shaft of the motor (2) is upwards connected with one end of a first rotating shaft (3) in the vertical direction, the other end of the first rotating shaft (3) is coaxially connected with a first disc (17), a first gear (4) is arranged on the first rotating shaft (3), a second rotating shaft (5) in the vertical direction is arranged at the bottom of the inner wall of the box body (1), one end of the second rotating shaft (5) is rotationally connected with the inner wall of the box body (1), a second disc (6) is arranged at the other end of the second rotating shaft (5), a second gear (7) is arranged on the second rotating shaft (5), the first gear (4) is equal to and meshed with the second gear (7) in diameter, a supporting plate (8) in the horizontal direction is arranged in the box body (1), and the first rotating shaft (3) and the second rotating shaft (5) both penetrate through the supporting plate (8), and is rotationally connected with the supporting plate (8), wherein the supporting plate (8) is positioned between the first gear (4) and the first disc (17);
the diameter of the first disc (17) is equal to that of the second disc (6), a first eccentric column (9) is arranged on the first disc (17), a second eccentric column (10) is arranged on the second disc (6), the first eccentric column (9) and the second eccentric column (10) are symmetrically arranged, the top end of the first eccentric column (9) is rotatably connected with a first sliding block (11), the upper end of the second eccentric column (10) is provided with a connecting rod (12), one end of the connecting rod (12) is rotatably connected with the upper end of the second eccentric column (10), the other end of the connecting rod (12) penetrates through the first sliding block (11) and is slidably connected with the first sliding block (11), the top end of the first sliding block (11) is provided with a guide port (13) in the horizontal direction, one end of a connecting column (14) is connected with the connecting rod (12), and the other end of the connecting column (14) penetrates through the guide port (13) and is connected with a workbench (15), the top end of the workbench (15) is provided with a plurality of placing grooves (16).
5. The preparation method of the inactivated vaccine adjuvant of the bacterial lysate according to claim 4, wherein the top end of the workbench (15) is provided with a cover (18), and the cover (18) is in threaded connection with the workbench (15).
6. The preparation method of the inactivated vaccine adjuvant of the bacterial lysate according to claim 4, wherein a limit block (19) is arranged at one end of the connecting rod (12) far away from the second eccentric column (10).
7. The method for preparing the inactivated vaccine adjuvant by using the bacterial lysate according to claim 4, wherein a box door (20) is arranged at the top end of the box body (1), and one end of the box door (20) is hinged with the top end of the box body (1).
8. The method for preparing the inactivated vaccine adjuvant by using the bacterial lysate according to claim 4, wherein a plurality of heat dissipation holes (21) are formed in the side wall of the box body (1).
9. The method for preparing inactivated vaccine adjuvant from bacterial lysate according to claim 4, wherein the high pressure homogenizer comprises: a cylinder body (22), a push plate (23), a fixed pipe (24), a first connecting rod (25), an opening (26), a sliding pipe (27), a second connecting rod (28) and a third connecting rod (29),
an electric push rod is arranged at the top of the inner wall of the barrel body (22), the output end of the electric push rod is downwards connected with a push plate (23), a fixed pipe (24) in the vertical direction is arranged below the push plate (23), the outer wall of the fixed pipe (24) is connected with the inner wall of the barrel body (22) through a first connecting rod (25), openings (26) in the vertical direction are respectively arranged at the left side and the right side of the outer wall of the fixed pipe (24), a sliding pipe (27) is arranged in the fixed pipe (24), the sliding pipe (27) can reciprocate up and down in the fixed pipe (24), second connecting rods (28) in the horizontal direction are arranged at the two ends of the sliding pipe (27), one end of each second connecting rod (28) is connected with the outer wall of the corresponding sliding pipe (27), and the other end of each second connecting rod (28) penetrates through the opening (26) to be vertically connected with one end of a third connecting rod (29), the other end of the third connecting rod (29) is connected with the bottom end of the push plate (23);
a rotating pipe (30) is arranged in the sliding pipe (27), a spiral sliding groove (31) is formed in the outer wall of the rotating pipe (30), a sliding column (32) is arranged on the inner wall of the rotating pipe (30), the sliding column (32) can slide in the spiral sliding groove (31), the bottom end of the rotating pipe (30) extends out of the sliding pipe (27) to be connected with a rotating plate (33), a first annular sliding groove (34) and a second annular sliding groove (35) are arranged on the inner wall of the lower end of the fixed pipe (24) at intervals respectively, the first annular sliding groove (34) is communicated with the second annular sliding groove (35) through a vertical groove (36), second sliding blocks (37) are arranged on the left side and the right side of the rotating plate (33) respectively, the second sliding blocks (37) can slide up and down in the vertical groove (36) in a reciprocating manner and can also rotate in the first annular sliding groove (34) and the second annular sliding groove (35), the below of fixed pipe (24) is provided with baffle (38) of horizontal direction, baffle (38) with barrel (22) inner wall connection, be provided with shaft hole (39) on baffle (38), the bottom and the third pivot (40) one end of commentaries on classics board (33) are connected, and third pivot (40) other end passes shaft hole (39) are connected with cutting knife (41), be provided with feed liquor pipe (42) and drain pipe (43) on the lower extreme outer wall of barrel (22) respectively.
10. The method for preparing the inactivated vaccine adjuvant by using the bacterial lysate according to claim 9, wherein the liquid inlet pipe (42) and the liquid outlet pipe (43) are both provided with high pressure valves (44).
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