Method for collecting protein and virus in mosquito saliva

Abstract

The invention discloses a method for collecting mosquito saliva protein and viruses. The invention provides a method for collecting protein and / or virus in saliva of mosquitoes. The method comprises the following steps: collecting saliva, separating saliva, washing, and collecting protein and / or virus. According to the method provided by the invention, a relatively large amount of mosquito salivary protein can be collected in a short time without causing any harm to mosquitoes, the mosquito salivary protein of the same mosquito can be collected at multiple time points, the utilization rate of the mosquitoes is remarkably improved, dynamic collection can be realized, special materials or equipment are not needed, and the cost is low. Therefore, the applicability is further improved.

Description

Technical Field

[0001] The invention relates to a method for collecting mosquito saliva proteins and viruses, belonging to the field of biotechnology. Background Art

[0002] Mosquito saliva contains over 100 proteins, and these mosquito salivary proteins are crucial for the feeding process (Reference 1). Numerous studies have demonstrated that mosquito salivary proteins have physiological functions such as lubricating mouthparts, anticoagulation, vasodilation, analgesia, and promoting initial food digestion (References 1-3). As important mediators of the interaction between mosquitoes and mammalian hosts, mosquito salivary proteins significantly influence host innate immunity, adaptive immunity, and hypersensitivity responses (References 1, 4, 5). More importantly, mosquito salivary proteins can significantly promote the transmission of mosquito-borne viruses (References 6, 7). Therefore, mosquito salivary proteins have become a research hotspot in fields such as immunology, vector biology, and mosquito-borne virology.

[0003] Because mosquitoes secrete only a small amount of mosquito salivary proteins, obtaining sufficient amounts for research is difficult, a key constraint in this area (references 8 and 9). Currently, the most widely used collection method is forced salivation (FS) (references 10-14). This method involves removing the mosquito's wings and legs and inserting its mouthparts into immersion oil in a capillary tube. After the mosquito secretes saliva into the immersion oil, the mosquito salivary proteins are extracted from the oil using PBS. The FS method is time-consuming and labor-intensive, and mosquitoes often die during the saliva collection process. Furthermore, the immersion oil occasionally leaks, making it difficult to completely recover it from the capillary tube, reducing the amount of mosquito salivary proteins collected. Furthermore, because this method only allows for a single collection of mosquito saliva, it is not possible to dynamically collect mosquito saliva from the same mosquito. Finally, certain components in the immersion oil may denature mosquito salivary proteins and inactivate mosquito-borne viruses. Some studies have reported that mosquito salivary proteins and viruses obtained through forced salivation differ in composition and infectivity from those secreted under physiological conditions (references 15, 16). These inherent defects limit the applicability of the FS method.

[0004] Therefore, there is an urgent need to develop new methods for collecting mosquito salivary proteins and viruses.

[0005] Citations

[0006] Reference 1 ：Wang, Z.Y., Nie, K.X., Niu, J.C., and Cheng, G. (2024). Research progress toward the influence of mosquito salivary proteins on the transmission of mosquito-borne viruses. Insect Sci 31, 663-673. 10.1111 / 1744-7917.13193.

[0007] Citation 2: Barillas-Mury, C., Ribeiro, J.M.C., and Valenzuela, J.G. (2022). Understanding pathogen survival and transmission by arthropod vectors to prevent human disease. Science 377, eabc2757. 10.1126 / science.abc2757.

[0008] Citation 3: Smith, L.B., Duge, E., Valenzuela-León, P.C., Brooks, S., Martin-Martin, I., Ackerman, H., and Calvo, E. (2022). Novel salivary antihemostatic activities of long-form D\(_7\) proteins from the malaria vector Anopheles gambiae facilitate hematophagy. J Biol Chem 298, 101971. 10.1016 / j.jbc.2022.101971.

[0009] Table 4: Martin-Martin, I., Valenzuela Leon, PC, Amo, L., Shrivastava, G., Iniguez, E., Aryan, A., Brooks, S., Kojin, BB, Williams, AE, Bolland, S., et al.(2022).Aedes aegypti facilitates sialokinin Mosquito blood feeding and modulates host immunity and vascular biology.Cell Rep 39,110648.10.1016 / j.cellrep.2022.110648.

[0010] Publication 5:Peng,Z.,and Simons,FE(2007).Advances in mosquito allergy.Curr Opin Allergy Clin Immunol 7,350-354.10.1097 / ACI.0b013e328259c313.

[0011] Figure 6:Wang, Z., Nie, K., Liang, Y., Niu, J., Yu, X., Zhang, O., Liu, L., Shi, X., Wang, Y., Feng, X., et al.(2024).A mosquito salivary protein-driven influx ofmyeloid cells facilitates flavivirus transmission.Embo j 43.1690–1721.10.1038 / s44318-024-00056-x.

[0012] Table 7: Sun, P., Nie, K., Zhu, Y., Liu, Y., Wu, P., Liu, Z., Du, S., Fan, H., Chen, CH, Zhang, R., et al.(2020).A mosquito salivary protein promotes flavivirus transmission by activation of autophagy.Nat Commun http: / / dx.doi.org / 10.1111 / j.1467-019-14115-z.

[0013] Annu RevEntomol 48,73-88.10.1146 / annurev.ento.48.060402.102812.Annu RevEntomol 48,73-88.10.1146 / annurev.ento.48.060402.102812.

[0014] Page 9:Fontaine, A., Pascual, A., Diouf, I., Bakkali, N., Bourdon, S., Fusai, T., Rogier, C., and Almeras, L. (2011). 4.33.10.1186 / 1756-3305-4-33.

[0015] Publication 10:Lefteri,DA,Bryden,SR,Pingen,M.,Terry,S.,McCafferty,A.,Beswick,EF,Georgiev,G.,Van der Laan,M.,Mastrullo,V.,Campagnolo,P.,et al.(2022).Mosquito saliva enhances virus infection through sialokinin-dependentvascular leakage.Proc Natl Acad Sci USA 119,e2114309119.10.1073 / pnas.2114309119.

[0016] Citation 11: Vogt, M. B., Lahon, A., Arya, R. P., Kneubehl, A. R., Spencer Clinton, J. L., Paust, S., and Rico-Hesse, R. (2018). Mosquito saliva alone has profound effects on the human immune system. PLoS Negl Trop Dis 12, e0006439. 10.1371 / journal.pntd.0006439.

[0017] Citation 12: Maharaj, P. D., Widen, S. G., Huang, J., Wood, T. G., and Thangamani, S. (2015). Discovery of mosquito saliva microRNAs during CHIKV infection. PLoS Negl Trop Dis 9, e0003386. 10.1371 / journal.pntd.0003386.

[0018] Citation 13: Smith, D. R., Carrara, A. S., Aguilar, P. V., and Weaver, S. C. (2005). Evaluation of methods to assess transmission potential of Venezuelan equine encephalitis virus by mosquitoes and estimation of mosquito saliva titers. Am J Trop Med Hyg 73, 33-39.

[0019] Cited literature 14: Conway, MJ, Watson, AM, Colpitts, TM, Dragovic, SM, Li, Z., Wang, P., Feitosa, F., Shepherd, DT, Ryman, KD, Klimstra, WB, et al. (2014). Mosquito saliva serine protease enhances dissemination of dengue virus into the mammalian host. J Virol 88,164-175.10.1128 / jvi.02235-13.

[0020] Cited literature 15: Styer, LM, Kent, KA, Albright, RG, Bennett, CJ, Kramer, LD, and Bernard, KA (2007). Mosquitoes inoculate high doses of West Nilevirus as they probe and feed on live hosts. PLoS Pathog 3, 1262-1270.10.1371 / journal.ppat.0030132.

[0021] Cited literature 16: Gloria-Soria, A., Brackney, DE, and Armstrong, PM (2022). Saliva collection via capillary method may underestimate arboviral transmission by mosquitoes. Parasit Vectors 15, 103.10.1186 / s13071-022-05198-7. Summary of the Invention

[0022] Problems to be solved by the invention

[0023] As previously mentioned, the FS method is time-consuming and prone to mosquito mortality, oil leakage, and residue during saliva collection, resulting in low mosquito saliva protein collection efficiency. Furthermore, the removal of wings and legs kills the mosquitoes, making them impossible to reuse and negating their potential for recycling.

[0024] In view of this, in order to solve the above-mentioned shortcomings of the prior art, such as the FS method, the present invention has developed a more efficient and convenient method for collecting mosquito salivary proteins. The present invention names this new method the Collecting-by-natural feeding (CNF) method, which can collect mosquito salivary proteins during natural feeding. The CNF method can collect a relatively large amount of mosquito salivary proteins in a short period of time without causing any harm to the mosquitoes, and can collect mosquito salivary proteins from the same mosquito at multiple time points, significantly improving the utilization rate of mosquitoes, and can be collected dynamically. It is worth noting that the CNF method does not require special materials or equipment, which further improves its applicability.

[0025] Solutions for solving problems

[0026] The present invention provides a method for collecting proteins and / or viruses in mosquito saliva, the method comprising:

[0027] a step of collecting saliva, wherein the mosquitoes whose saliva is to be collected for protein and / or virus are sucked on mosquito liquid food in a collecting component and secrete saliva into the collecting component, thereby obtaining mosquito liquid food containing saliva in the collecting component;

[0028] a step of separating saliva, wherein the mosquito liquid food containing saliva in the collecting part is separated from the collecting part by centrifugation to obtain a first filtrate;

[0029] a washing step, wherein the collecting component after centrifugation in the saliva separation step is soaked in a cleaning solution, and then the remaining saliva in the collecting component is separated from the collecting component by squeezing and / or centrifugation to obtain a second filtrate;

[0030] The step of collecting proteins and / or viruses comprises mixing the first filtrate and the second filtrate, and concentrating the mixture to obtain proteins and / or viruses in mosquito saliva.

[0031] In some embodiments, the cleaning solution comprises a solution of a weak acid and its conjugate acid salt.

[0032] In some preferred embodiments, the cleaning solution comprises at least one of a phosphate buffer, an acetate buffer, and a carbonate buffer.

[0033] In some more preferred embodiments, the cleaning solution is phosphate buffered saline.

[0034] In some embodiments, in the steps of separating the saliva and washing, the centrifugation is performed at a temperature not higher than 10°C.

[0035] In some preferred embodiments, the centrifugation is performed at a temperature of 2-6°C.

[0036] In some embodiments, before the step of collecting saliva, the mosquitoes from which proteins and / or viruses in saliva are to be collected are fasted.

[0037] In some preferred embodiments, the fasting treatment time is within 72 hours.

[0038] In some embodiments, the mosquito liquid food comprises a carbohydrate solution.

[0039] In some preferred embodiments, the mosquito liquid food comprises at least one of a glucose solution, a maltose solution, a sucrose solution, and a lactose solution.

[0040] In some more preferred embodiments, the carbohydrate solution comprises a sucrose solution.

[0041] In some embodiments, the concentration of the sucrose solution is 5-20 (w / v)%.

[0042] In some preferred embodiments, the concentration of the sucrose solution is 8-15 (w / v)%.

[0043] In some embodiments, in the step of collecting saliva, the mosquitoes whose saliva is to be collected for protein and / or virus ingest the mosquito liquid food in the collecting component under light-proof conditions.

[0044] In some embodiments, in the step of collecting saliva, the mosquitoes whose saliva is to be collected for protein and / or virus feed on the mosquito liquid food in the collecting component for 1 to 3 hours in a dark condition.

[0045] In some embodiments, in the step of collecting saliva, the collecting component is made of a material that is water-absorbent and does not contain biological protein components.

[0046] In some preferred embodiments, the collecting member is a sponge.

[0047] In some more preferred embodiments, the collecting member is a sponge composed of at least one of polyurethane, polyvinyl alcohol, or polyglycolic acid.

[0048] In some preferred embodiments, in the step of collecting proteins and / or viruses, the concentration is performed using an ultrafiltration centrifuge tube.

[0049] Effects of the Invention

[0050] (1) The present invention has developed a more efficient and convenient method for collecting mosquito salivary proteins. This new method is named the Collecting-by-natural feeding (CNF) method because it can collect mosquito salivary proteins during natural feeding. The CNF method can collect a relatively large amount of mosquito salivary proteins in a short period of time without causing any harm to the mosquitoes, and can collect mosquito salivary proteins from the same mosquito at multiple time points, significantly improving the utilization rate of mosquitoes, and can be collected dynamically. It is worth noting that the CNF method does not require special materials or equipment, which further improves its applicability.

[0051] (2) The CNF method is simple to operate and can collect significantly more mosquito salivary proteins in a short period of time without causing any harm to the mosquitoes. It supports the dynamic collection of mosquito salivary proteins and the reuse of mosquitoes.

[0052] (3) Compared with the FS method, the CNF method has unique advantages in terms of ease of operation, time saving, reusable mosquitoes, and greater biological activity of collected salivary proteins and viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the FS (Forced salivation) method and the CNF (Collecting-by-natural feeding) method. Figure 1 A and B in the figure are schematic diagrams of the FS method. Figure 1 C and D in FIG are schematic diagrams of the CNF method.

[0054] Figure 2 A comparison chart of the convenience and efficiency of the FS method and the CNF method.

[0055] Figure 2 A in the equation is the time required to collect salivary proteins from 100 mosquitoes using the two methods. Figure 2 B is the amount of mosquito salivary protein collected from each mosquito by the two methods. Three independent operators strictly followed the steps of the FS and CNF methods, and the operation time and amount of mosquito salivary protein (MSP) collected by each operator were recorded.

[0056] Figure 3 Comparison of the biological activities of mosquito salivary proteins and viruses in mosquito saliva collected by FS and CNF methods.

[0057] Figure 3 AC are the biological activities of mosquito salivary proteins collected by two methods.

[0058] Figure 3A in the figure is the ability to stimulate RAW264.7 cells to produce IL-1β to quantify its biological activity. Figure 3 B in the figure is the ability to stimulate RAW264.7 cells to produce IL-6 to quantify its biological activity and Figure 3 C in the figure quantifies its biological activity by stimulating RAW264.7 cells to produce TNF-α.

[0059] Figure 3 D in the figure is the titer of DENV-2 in mosquito saliva collected by two methods. DETAILED DESCRIPTION

[0060] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0061] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0062] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0063] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0064] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in various embodiments in any appropriate manner.

[0065] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B. Detailed Description of the Invention

[0067] Method for collecting proteins and / or viruses from mosquito saliva

[0068] The present invention provides a method for collecting proteins and / or viruses in mosquito saliva, the method comprising:

[0069] a step of collecting saliva, wherein the mosquitoes whose saliva is to be collected for protein and / or virus are sucked on mosquito liquid food in a collecting component and secrete saliva into the collecting component, thereby obtaining mosquito liquid food containing saliva in the collecting component;

[0070] a step of separating saliva, wherein the mosquito liquid food containing saliva in the collecting part is separated from the collecting part by centrifugation to obtain a first filtrate;

[0071] a washing step, wherein the collecting component after centrifugation in the saliva separation step is soaked in a cleaning solution, and then the remaining saliva in the collecting component is separated from the collecting component by squeezing and / or centrifugation to obtain a second filtrate;

[0072] The step of collecting proteins and / or viruses comprises mixing the first filtrate and the second filtrate, and concentrating the mixture to obtain proteins and / or viruses in mosquito saliva.

[0073] Steps for collecting saliva

[0074] In some embodiments, mosquitoes whose saliva proteins and / or viruses are to be collected are allowed to suck mosquito liquid food in a collecting component and secrete saliva into the collecting component, thereby obtaining mosquito liquid food containing saliva in the collecting component.

[0075] (mosquito)

[0076] The mosquito described in the present invention is an insect with piercing-sucking mouthparts, which are insects that feed on plant sap or animal blood and can both pierce the host body and suck the host body fluid. The lower lip of the piercing-sucking mouthparts is extended into a beak, and the upper and lower jaws are specialized into needles, which are suitable for piercing animal and plant tissues and sucking blood and cell fluid. It includes insects of the orders Homoptera, Hemiptera, Siphonaptera, Pseudotoptera and Diptera.

[0077] In some embodiments, the mosquito of the present invention is an insect of the family Culicidae of the order Diptera.

[0078] In some embodiments, the mosquitoes described herein include adult mosquitoes.

[0079] In some embodiments of the present invention, the mosquito comprises one or more of Culex, Culiseta, Anopheles and Aedes.

[0080] In some exemplary embodiments, the mosquito comprises Aedes aegypti.

[0081] (Collecting Parts)

[0082] In some embodiments, the step of collecting saliva uses a collecting component to collect mosquito saliva.

[0083] In some embodiments, the collecting component can retain liquids such as mosquito liquid food and mosquito saliva, and can separate liquids such as mosquito liquid food and mosquito saliva from the collecting component by means such as centrifugation. For example, a water-absorbent material and / or a porous material can be selected.

[0084] In some preferred embodiments, the collecting component is made of a material having good water absorption and containing no biological protein components.

[0085] In some preferred embodiments, the collecting component has been sterilized before use.

[0086] Sterile processing and materials that do not contain biological protein components can minimize the risk of exogenous protein contamination during the collection of mosquito salivary protein (MSP).

[0087] In some more preferred embodiments, the collecting component is a synthetic polymer sponge (hereinafter referred to as sponge), for example, a sponge composed of at least one of polyurethane (PU), polyvinyl alcohol (PVA) or polyglycolic acid (PGA). Further preferably, the collecting component is made of polyurethane foam.

[0088] It can be understood that the size of the collecting component can be selected according to actual needs.

[0089] In some exemplary embodiments, the specific parameters of the size of the collecting component are 1.5×1.5×1.0 cm 3 (length × width × height), a sterile synthetic polymer sponge made of protein-free polyurethane foam.

[0090] In some preferred embodiments, the sponge contains mosquito liquid food before collecting saliva.

[0091] In some embodiments, during the saliva collection step, the mosquitoes whose saliva is to be collected for proteins and / or viruses can be placed in a culture container (e.g., a mosquito cup), which is provided with a mesh cover, for example, which can be installed at the opening of the culture container and detachably connected to the culture container. The mesh cover is used to open or close the opening at the top of the culture container. The collecting component can be placed on the mesh cover, which keeps the mosquitoes whose saliva is to be collected for proteins and / or viruses in the culture container in the culture container, and can feed on the mosquito liquid food in the collecting component through the pores of the mesh cover, while secreting its saliva into the collecting component, that is, the mosquito saliva is collected in the collecting component through the mosquito feeding.

[0092] The present invention does not impose any particular limitation on the above-mentioned culture container, as long as mosquitoes can survive in the culture container.

[0093] The present invention does not impose any particular limitation on the mesh cover, as long as the mosquitoes can complete feeding through the pores of the mesh cover and the mosquitoes are kept in the culture container.

[0094] In some exemplary embodiments, the above-mentioned culture container, mesh cover and collection component can refer to Figure 1 D in.

[0095] (Liquid mosquito food)

[0096] In the present invention, there is no particular limitation on the mosquito liquid food, as long as it can be retained by and separated from the collecting component, can be eaten by mosquitoes, and does not affect the subsequent acquisition of salivary proteins and / or viruses and their activity.

[0097] In some embodiments, the mosquito liquid food comprises at least one of plant juice and a carbohydrate solution.

[0098] In some preferred embodiments, the mosquito liquid food comprises a carbohydrate solution.

[0099] In some embodiments, the carbohydrate compound is polyhydroxy aldehyde, polyhydroxy ketone, and an organic compound that can be hydrolyzed to generate polyhydroxy aldehyde or polyhydroxy ketone, which can be divided into monosaccharides, disaccharides, and polysaccharides.

[0100] In some embodiments, the carbohydrate compound may be at least one of glucose, maltose, sucrose, and lactose.

[0101] In some specific embodiments, the carbohydrate compound solution may be a solution containing sucrose.

[0102] In some embodiments, the concentration of the sucrose solution is 5-20 (w / v)%, preferably 8-15 (w / v)%, more preferably 9-12 (w / v)%, for example 9%, 10%, 11%, 12%.

[0103] In some specific embodiments, the solvent of the sucrose solution is water.

[0104] In some exemplary embodiments, the collecting component (eg, sponge) contains a 10% (w / v) sucrose solution, and when the mosquito feeds, the collecting component (eg, sponge) absorbs the mosquito saliva.

[0105] In some exemplary embodiments, Figure 1 As shown in D in the figure, mosquitoes can survive normally in the mosquito cup. Mosquitoes whose saliva proteins and / or viruses are to be collected are placed in the mosquito cup. The mosquitoes whose saliva proteins and / or viruses are to be collected feed at the pores of the mesh cover above the mosquito cup. The sponge is placed above the mesh cover. The mosquitoes suck the sucrose solution in the sponge and release saliva, and the sponge absorbs the mosquito saliva.

[0106] (Pre-eating preparation)

[0107] In some embodiments, before the step of collecting saliva, the mosquitoes whose saliva is to be collected for protein and / or virus need to be subjected to pre-feeding treatment.

[0108] In some embodiments, pre-feeding treatment comprises fasting treatment.

[0109] In some preferred embodiments, the fasting treatment is within about 72 hours, more preferably, within about 48 hours, and even more preferably, more than 12 hours or more than 24 hours. Mosquitoes can increase saliva secretion when feeding after fasting treatment.

[0110] (Processing during eating)

[0111] In some embodiments, in the step of collecting saliva, the mosquitoes whose saliva is to be collected for protein and / or virus ingest the mosquito liquid food in the collecting component under quiet, light-proof conditions (dark environment).

[0112] In some exemplary embodiments, the ambient noise is lower than 55 decibels, preferably, the ambient noise is lower than 50 decibels, and more preferably, the ambient noise is lower than 40 decibels.

[0113] Reducing environmental noise can reduce interference with mosquito feeding, thereby facilitating the collection of saliva.

[0114] The present invention does not specify the method of avoiding light, which can be a commonly used method by those skilled in the art. In some preferred embodiments, avoiding light can be that there is no light source in the environment when the mosquitoes feed.

[0115] In some exemplary embodiments, the light-shielding treatment may include turning off the lights in a laboratory where mosquitoes are fed.

[0116] Mosquitoes rely primarily on their sense of smell (smell) and close-up taste to locate sugar sources. Avoiding light can reduce the impact of other interference sources on the mosquito feeding process, further promoting the secretion of saliva.

[0117] Steps for separating saliva

[0118] In some embodiments, the mosquito liquid food containing saliva in the collecting component is separated from the collecting component by centrifugation to obtain a first filtrate (hereinafter also referred to as filtrate 1).

[0119] In some embodiments, the centrifugation includes centrifuging a collecting component to separate the mosquito liquid food containing saliva from the collecting component to obtain a first filtrate containing mosquito saliva and mosquito liquid food.

[0120] In some specific embodiments, such as Figure 1 As shown in c, the centrifugation includes placing a collection component (such as a sponge) containing mosquito saliva in the upper chamber of a centrifuge tube with a diaphragm, performing centrifugation, and collecting a first filtrate of mosquito liquid food containing saliva from the lower chamber of the centrifuge tube.

[0121] In some preferred embodiments, the centrifugation is performed at a temperature not higher than 10°C. More preferably, the centrifugation is performed at a temperature between 2-6°C, such as 3°C, 4°C, 5°C, etc.

[0122] In some preferred embodiments, the centrifugation is performed at a centrifugal force of no higher than 15,000 g. More preferably, the centrifugal force is between 9,000 g and 13,000 g, such as 9500 g, 10,000 g, 11,000 g, and the like.

[0123] In some preferred embodiments, the centrifugation time does not exceed 6 min. More preferably, the centrifugation time is 2-5 min, such as 2.5 min, 3 min, 3.5 min, etc.

[0124] In some exemplary embodiments, the centrifugation parameters may be 10,000 g at 4° C. for 3 minutes.

[0125] Washing steps

[0126] In some embodiments, the collecting component after centrifugation in the saliva separation step is soaked in a cleaning solution, and then the remaining saliva in the collecting component is separated from the collecting component by squeezing and / or centrifugation to obtain a second filtrate (hereinafter also referred to as filtrate 2).

[0127] In some embodiments, in the washing step, the remaining mosquito saliva is released from the collecting component by squeezing and / or centrifuging to obtain a second filtrate containing the remaining mosquito saliva in the collecting component.

[0128] In some embodiments, a cleaning solution is used to soak the collection component after centrifugation in the saliva separation step, and the remaining saliva in the collection component is separated from the collection component by squeezing and further centrifugation to obtain a second filtrate.

[0129] In some embodiments, the cleaning solution comprises a solution composed of a weak acid and its conjugate acid salt. Preferably, the cleaning solution comprises at least one of a phosphate buffer, an acetate buffer and a carbonate buffer. More preferably, the cleaning solution is a phosphate buffer. Further preferably, the cleaning solution is PBS.

[0130] In some embodiments, a cleaning solution (such as PBS) is added to the collecting component after the saliva separation step, the collecting component is soaked with the cleaning solution (such as PBS), and the collecting component is squeezed to release the remaining mosquito saliva in the collecting component.

[0131] In some embodiments, the pH of the cleaning solution is between 6.0 and 7.5. Preferably, the pH of the cleaning solution is between 7.0 and 7.5. More preferably, the pH of the cleaning solution (eg, PBS) is 7.4.

[0132] In some embodiments, the temperature of the cleaning solution is not higher than 10°C, preferably, the temperature of the cleaning solution is not higher than 5°C, and more preferably, the temperature of the cleaning solution (such as PBS) is 4°C.

[0133] In some embodiments, 4° C. PBS is added to the collection component after the saliva separation step to soak the collection component. Those skilled in the art can select the volume of the soaking liquid within a reasonable range according to the size of the collection component within the scope provided by the present invention.

[0134] In some embodiments, the present invention is in the form of a 1.5×1.5×1.0 cm 3 A collection component (such as a sponge) is added with a volume of 1-5 mL of a 4°C PBS solution. Preferably, the volume of the PBS solution is 1.5-3 mL, and more preferably, the volume of the PBS solution is 2 mL.

[0135] In some exemplary embodiments, a collection member (eg, a sponge) soaked in a soaking liquid is squeezed using tweezers to release mosquito saliva from the collection member (eg, a sponge).

[0136] In some embodiments, after squeezing the collecting component to release the remaining mosquito saliva in the collecting component, the collecting component is further centrifuged to separate the remaining mosquito saliva in the collecting component from the collecting component to obtain a second filtrate containing the remaining mosquito saliva in the collecting component.

[0137] In some specific embodiments, the centrifugation includes placing a collection component (such as a sponge) containing mosquito saliva in the upper chamber of a centrifuge tube with a diaphragm, performing centrifugation, and collecting a second filtrate containing mosquito saliva from the lower chamber of the centrifuge tube.

[0138] It can be understood that the second filtrate contains the cleaning liquid (separated from the collecting component during squeezing and centrifugation of the collecting component), the mosquito liquid food remaining in the collecting component after the saliva separation step, and the mosquito saliva (and the proteins and / or viruses in the saliva).

[0139] In some preferred embodiments, the centrifugation is carried out at a temperature not higher than 10°C. More preferably, the centrifugation is carried out at a temperature between 2°C and 6°C, such as 3°C, 4°C, 5°C, etc.

[0140] In some preferred embodiments, the centrifugation is performed at a centrifugal force of no more than 15,000 g. More preferably, the centrifugal force is between 9,000 g and 13,000 g, such as 9500 g, 10,000 g, 11,000 g, and the like.

[0141] In some preferred embodiments, the centrifugation time does not exceed 6 min. More preferably, the centrifugation time is 2-5 min, such as 2.5 min, 3 min, 3.5 min, etc.

[0142] In some exemplary embodiments, the centrifugation parameters may be consistent with the centrifugation parameters in the step of separating saliva, for example, centrifugation at 10,000 g for 3 minutes at 4°C.

[0143] In some embodiments, the washing step may be repeated at least once to completely release the mosquito saliva remaining in the collecting component to obtain a filtrate.

[0144] Steps for collecting proteins and / or viruses

[0145] In some embodiments, the mosquito saliva contains mosquito salivary proteins and / or viruses.

[0146] In some embodiments, the step of collecting proteins and / or viruses comprises mixing the first filtrate and the second filtrate, and obtaining the proteins and / or viruses in the mosquito saliva by concentration.

[0147] In some embodiments, the first filtrate and the second filtrate are subjected to ultrafiltration and / or concentration.

[0148] The present invention does not specify ultrafiltration and concentration treatments, which may be commonly used by those skilled in the art. In some exemplary embodiments, the first filtrate and the second filtrate are combined, and the combined filtrate is concentrated using an ultrafiltration centrifuge tube.

[0149] The molecular weight cut-off of the ultrafiltration centrifuge tube can be selected according to actual needs. For example, to minimize the loss of salivary proteins and retain more abundant salivary proteins, an ultrafiltration centrifuge tube with a molecular weight cut-off of 3kDa can be selected. Using an ultrafiltration centrifuge tube with a molecular weight cut-off of more than 3kDa (e.g., 10kDa) will result in the loss of some salivary proteins.

[0150] There is no particular limitation on the material of the ultrafiltration membrane of the ultrafiltration centrifuge tube. In some preferred embodiments, a regenerated cellulose membrane can be selected.

[0151] In some exemplary embodiments, the collected filtrate 1 and filtrate 2 are combined, concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and replaced with a washing solution (eg, PBS) to obtain PBS containing mosquito saliva.

[0152] In some embodiments, the replacement includes centrifuging the liquid filtered by the ultrafiltration tube at least twice, retaining the upper layer of liquid during the first centrifugation; the second centrifugation includes adding a new washing solution (such as PBS) to the upper layer of liquid retained after the first centrifugation, washing it, and then centrifuging to retain the upper layer of liquid, thereby completing one replacement; the third centrifugation includes adding a new washing solution (such as PBS) to the upper layer of liquid retained after the second centrifugation, washing it, and then centrifuging to retain the upper layer of liquid, thereby completing a second replacement, and mosquito saliva is obtained after the second replacement.

[0153] In some embodiments, the replacement is performed at a temperature not higher than 10°C. Preferably, the replacement is performed at a temperature between 2°C and 6°C, such as 3°C, 4°C, 5°C, etc.

[0154] In some embodiments, the first centrifugation during the replacement is performed at no more than 8000 rpm. Preferably, the centrifugation is performed at 3000 rpm-6500 rpm. More preferably, the centrifugation is performed at 4500 rpm-5500 rpm, for example, 4500 rpm, 5000 rpm, 5500 rpm, etc.

[0155] In some embodiments, the first centrifugation time during the replacement is no less than 60 min. Preferably, the centrifugation time is between 15-50 min. More preferably, the centrifugation time is between 20-40 min, such as 25 min, 30 min, 35 min, etc.

[0156] In some embodiments, the second centrifugation during the replacement is performed at no more than 8000 rpm. Preferably, the centrifugation is performed at 3000 rpm-6500 rpm. More preferably, the centrifugation is performed at 4500 rpm-5500 rpm, for example, 4500 rpm, 5000 rpm, 5500 rpm, etc.

[0157] In some embodiments, the second centrifugation time during the replacement is no less than 60 min. Preferably, the centrifugation time is between 15-50 min. More preferably, the centrifugation time is between 20-40 min, such as 25 min, 30 min, 35 min, etc.

[0158] In some embodiments, the third centrifugation during the replacement is performed at no more than 8000 rpm. Preferably, the centrifugation is performed at 3000 rpm-6500 rpm. More preferably, the centrifugation is performed at 4500 rpm-5500 rpm, for example, 4500 rpm, 5000 rpm, 5500 rpm, etc.

[0159] In some embodiments, the third centrifugation time during the replacement is no less than 80 min. Preferably, the centrifugation time is between 30-60 min. More preferably, the centrifugation time is between 35-55 min, such as 35 min, 40 min, 45 min, etc.

[0160] In some exemplary embodiments, the replacement is performed by centrifugation at 5,000 rpm for 30 minutes at 4°C in a flat rotor centrifuge, the lower layer is discarded, 15 mL of pre-cooled PBS is added to the upper layer, and the mixture is centrifuged at 5,000 rpm for 30 minutes at 4°C. The lower layer is then discarded, and 15 mL of pre-cooled PBS is added to the upper layer, and the mixture is centrifuged at 5,000 rpm for 40 minutes at 4°C.

[0161] In some preferred embodiments, the final concentrated volume of the mosquito saliva obtained after the secondary replacement is 200-500 μL, for example, the final concentrated volume is 200 μL, 300 μL, 400 μL, 500 μL, etc.

[0162] The present invention names this saliva collection method, Collecting-by-natural feeding (CNF), which allows mosquito salivary proteins to be collected during natural feeding. The CNF method can collect a relatively large amount of mosquito salivary proteins in a short period of time without causing any harm to the mosquitoes. It can also collect salivary proteins from the same mosquito at multiple time points, significantly improving the utilization rate of the mosquitoes and enabling dynamic collection.

[0163] In some embodiments, the present invention uses a Bradford-based assay to quantify the protein concentration of MSP.

[0164] In some exemplary embodiments, the present invention uses a Bradford protein quantification kit to quantify the protein concentration of MSP.

[0165] In some embodiments, the average time required to obtain mosquito saliva by the above method of the present invention is no more than 200 minutes. Preferably, the average time required to obtain mosquito saliva is no more than 190 minutes.

[0166] The average time consumption of mosquito saliva obtained by the method of the present invention is better than that of the traditional method (FS).

[0167] As used herein, "IL-1β" means interleukin-1β.

[0168] As used herein, "IL-6" refers to interleukin-6.

[0169] As used herein, "TNF-α" refers to interleukin-1 beta.

[0170] In some embodiments, treatment of mouse macrophage cell line RAW264.7 with 1 μg / mL of mosquito saliva protein obtained by the above-mentioned method of the present invention significantly increased the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α by the cells. Furthermore, the bioactivity of mosquito saliva protein collected by the CNF method provided by the present invention was significantly higher than that collected by the FS method.

[0171] In some embodiments, the virus is inoculated into mosquitoes, saliva is collected using the method of the present invention, and the virus content in the saliva is detected.

[0172] In some embodiments, the titer of the virus described herein is measured by plaque formation.

[0173] In some exemplary embodiments, dengue virus type 2 (DENV-2) is cultured in C6 / 36 cells, 100 PFU of DENV-2 is microinjected into the thorax of mosquitoes, mosquito saliva is collected by the CNF method described in the present invention, and the titer is determined on Vero cells by a plaque formation assay.

[0174] As used herein, "C6 / 36 cells" refers to a mosquito larval cell line derived from Aedes albopictus.

[0175] As used herein, "Vero cells" refers to a continuously cultured cell line derived from African green monkey kidney epithelial cells.

[0176] In some embodiments, the mosquito saliva virus titer obtained by the above method of the present invention is not less than 20 PFU / saliva sample, preferably, the virus titer is not less than 25 PFU / saliva sample. The mosquito saliva virus titer collected by the CNF method provided by the present invention is significantly higher than the mosquito saliva virus titer collected by the FS method.

[0177] Compared with the FS method, the CNF method provided by the present invention has advantages in terms of convenient operation, time saving, reusable mosquitoes, and biological activity of salivary proteins and viruses.

[0178] Example

[0179] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0180] Main Materials

[0181] Aedes aegypti (Rockefeller strain) were reared in a dedicated incubator (Cat# Model 818, Thermo Fisher) at 28°C and 80% humidity according to standard rearing procedures.

[0182] Example 1 CNF method

[0183] First, 100 Aedes aegypti (Rockefeller strain) were starved two days (48 hours) prior to collection. A commercially available medical-grade sponge (Cat# single use system, MUNKCARE) was used. The sponge was sterilized and made of protein-free polyurethane foam, minimizing the risk of exogenous protein contamination during MSP collection. The sponge dimensions were 1.5 × 1.5 × 1.0 cm.3 , soaked with 10% (w / v) sterile sucrose solution (Cat# S8271, Solarbio), the sponge filled with 10% (w / v) sucrose solution was placed on the mesh cover of a truncated cone-shaped mosquito cup (10 cm high, 10 cm upper bottom diameter, 7.5 cm lower bottom diameter), and the mosquito cup was placed in a dark, undisturbed environment (ambient noise below 40 decibels and no obvious light source) for about 1 hour to promote the natural feeding and saliva secretion of mosquitoes ( Figure 1 D) The mosquito sucks the sucrose water from the sponge and secretes saliva into the sponge.

[0184] Carefully grab the sponge and place it in the upper chamber of a 50mL centrifuge tube (Cat#CTF-50-NY-45-S, LABSELECT) with a septum (filter). Centrifuge at 10,000g for 3 minutes at 4°C. Collect the filtrate 1 containing mosquito salivary proteins from the lower chamber of the centrifuge tube. Add 2mL of 4°C pre-cooled PBS (pH=7.4) to soak the sponge. Squeeze the sponge with tweezers to completely release the mosquito salivary proteins. Under the same centrifugation conditions,

[0185] Repeat the centrifugation step and collect filtrate 2.

[0186] The collected filtrate 1 and filtrate 2 were combined and concentrated using a 3 kDa molecular weight cutoff ultrafiltration centrifuge tube (Cat# UFC9003, Millipore). Specifically, the filtrate was centrifuged at 5,000 rpm for 30 min at 4°C in a flat rotor centrifuge. The lower layer was discarded, and 15 mL of pre-cooled PBS was added to the upper layer and centrifuged at 5,000 rpm for 30 min at 4°C. The lower layer was discarded, and 15 mL of pre-cooled PBS was added to the upper layer and centrifuged at 5,000 rpm for 40 min at 4°C. The final concentrated volume was 200-500 μL ( Figure 1 C).

[0187] The protein concentration was measured (using the Bradford protein quantification kit based on the Bradford method (Cat# P0006, Beyotime) according to the manufacturer's instructions to quantify the MSP concentration, the same below) and the samples were frozen in liquid nitrogen or stored at -80°C.

[0188] Comparative Example 1 FS method

[0189] To collect MSP using the FS method, place Aedes aegypti mosquitoes on ice for 15 minutes, then remove their wings and legs. Insert the mouthparts of a single mosquito into a capillary tube or pipette tip pre-filled with 10 μL of immersion oil (Cat# I0765, Sigma Aldrich). A very small bubble of air can be seen in front of the mosquito's mouthpiece; this represents saliva secretion. Congestion of the mosquito's abdomen can also be used as an indicator of successful salivation, as the mosquito secretes saliva into the immersion oil during feeding. Allow the Aedes aegypti mosquitoes to salivate in the immersion oil for 1 hour at room temperature. After salivation is complete, carefully remove the mosquitoes and collect the immersion oil containing saliva into a 1.5 mL centrifuge tube. Add 4 mL of pre-chilled 100 μL of HCl to the collected oil (pH 7.4) and vortex thoroughly to mix. Incubate the mixture on ice for 10 minutes and then centrifuge at 10,000 g for 10 minutes at 4°C. The upper oil phase was discarded and the PBS containing mosquito saliva was collected ( Figure 1 A and B in the sample). After measuring the protein concentration using the same method as in Example 1, the sample was frozen in liquid nitrogen or stored at -80°C.

[0190] Test Case

[0191] Statistical analysis in all the following test cases used quantitative data that conformed to a normal distribution and had homogeneous variances and were expressed as mean ± standard error (SEM); otherwise, the data were expressed as median. Statistical analysis was performed using GraphPad Prism 10.0.0 software. Quantitative data between two groups were compared using an unpaired t-test (parametric test) or a Mann-Whitney test (nonparametric test). Statistical significance was set at p < 0.05. ns indicates no significant difference, * indicates p < 0.05, and **** indicates p < 0.0001.

[0192] Test Example 1: Comparison of Collection Time Between Two Methods

[0193] First, mosquito salivary proteins were collected from 100 mosquitoes using both the FS and CNF methods. Three independent operators strictly followed the FS and CNF procedures. The operation time and amount of mosquito salivary protein (MSP) collected were recorded for each operator.

[0194] The results showed that the FS method took an average of 284 minutes to collect salivary proteins from 100 mosquitoes ( Figure 2 A in the figure), while the CNF method takes an average of 184 minutes ( Figure 2 A) in the figure shows that the CNF method saves a lot of time compared to the FS method.

[0195] Test Example 2: Comparison of the concentration of mosquito saliva proteins collected by two methods

[0196] The concentration of MSP was quantified using a Bradford protein quantification kit (Cat# P0006, Beyotime) based on the Bradford method according to the manufacturer's instructions.

[0197] The average mosquito salivary protein collected by FS method was 23.65ng ( Figure 2 B), while the mosquito salivary protein collected by CNF method was 23.22ng ( Figure 2 B) in the figure shows that the concentrations of mosquito salivary proteins collected by the CNF method and the FS method are similar, but the CNF method takes less time and is more efficient.

[0198] Test Example 3 Comparison of the biological activity of mosquito salivary proteins in mosquito saliva collected by two methods

[0199] When the FS method is used to collect mosquito saliva, the proteins and viruses in the saliva are in direct contact with the immersion oil for more than 1 hour ( Figure 1 A in the middle), which is likely to affect the activity of proteins and viruses. In addition, several studies have reported (references: (1) Styer, LM, Kent, KA, Albright, RG, Bennett, CJ, Kramer, LD, and Bernard, KA (2007). Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts. PLoS Pathog 3, 1262-1270.10.1371 / journal.ppat.0030132.; (2) Gloria-Soria, A., Brackney, DE, and Armstrong, PM (2022). Saliva collection viacapillary method may underestimate arboviral transmission by mosquitoes. Parasit Vectors 15,103.10.1186 / s13071-022-05198-7.) The composition and viral titer of mosquito salivary proteins in mosquito saliva collected by the FS method differ significantly from those in saliva secreted by mosquitoes under physiological conditions. Next, the present invention compared the biological activity and viral titer of mosquito salivary proteins collected by the FS and CNF methods. It has been reported that mosquito salivary proteins can stimulate macrophages to express multiple cytokines.

[0200] The present invention uses 1 μg / mL mosquito saliva protein collected by the FS method or the CNF method to treat the mouse macrophage cell line RAW264.7 cells (purchased from ATCC, catalog number TIB-71). Mouse macrophage RAW264.7 cells (Cat#TIB-71, ATCC) are cultured at 37°C in RPMI 1640 medium (Cat#22400089, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Cat#ST30-3302, PAN) and 1% antibiotic-antimycotic drugs (Cat#15240-062, Invitrogen).

[0201] MSP biological activity detection method: RAW264.7 cells were plated at 1×10 4 Seed 10 cells / well in a 96-well plate. Carefully aspirate the remaining medium from each well before treatment. Add a mosquito saliva protein solution prepared in RPMI 1640 medium to the cells at a final concentration of 1 μg / mL, using a treatment volume of 100 μL / well. Return the cells to a 37°C, 5% CO2 incubator. PBS-treated cells served as a negative control. Treatment lasted for 4 hours, with four replicates.

[0202] After incubation, cells were washed with PBS, and total RNA was extracted using the AxyPrep™ Multisource Total RNA Miniprep Kit (Cat#AP-MN-MS-RNA-250, Axygen). Total RNA was reverse transcribed into total cDNA using the iScript cDNA Synthesis Kit (Cat#170-8890, Bio-Rad). RNA abundance of specific genes (IL-1β, IL-6, TNF-α, and GAPDH) was quantified using the iTaq Universal SYBR Green Supermix Kit (Cat#1725121, Bio-Rad) on a Bio-Rad CFX-96 Touch Real-Time Detection System. Mouse IL-1β, IL-6, and TNF-α mRNA expression was normalized to GAPDH. IL-1β, IL-6, and TNF-α mRNA expression was normalized to that of GAPDH.

[0203] Therefore, RAW264.7 cells were treated with the same concentration of mosquito saliva proteins collected by the two methods for 4 hours. The results showed that the expression of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α in RAW264.7 cells treated with mosquito saliva proteins collected by the CNF method was significantly increased compared with cells treated with mosquito saliva proteins collected by the FS method ( Figure 3 A- Figure 3C) indicates that the bioactivity of mosquito salivary proteins collected using the CNF method is significantly higher than that collected using the FS method. This may be because the CNF method collects mosquito salivary proteins under normal feeding conditions, which differs in type and quantity from the FS method, which collects mosquito salivary proteins under forced secretion conditions. Furthermore, certain components in the immersion oil during the FS method collection process may also affect the bioactivity of mosquito salivary proteins.

[0204] In summary, compared with the FS method, the CNF method can better preserve the biological activity of mosquito salivary proteins.

[0205] Test Example 4 Comparison of virus titers in mosquito saliva collected by two methods

[0206] In the study of vector biology and mosquito-borne viruses, the detection of virus titers in mosquito saliva is a routine and important task. The outermost layer of mosquito-borne viruses is a lipid envelope, and direct contact of virus particles with immersion oil may inactivate the virus. Next, the present invention compared the virus titers in mosquito saliva collected by two methods. First, the virus was used to infect mosquitoes. In order to infect Aedes aegypti, 100 PFU of DENV-2 was microinjected into the thorax of the mosquito. The virus-inoculated mosquitoes were maintained in a breeding climate chamber at 28°C for 8 days, after which saliva was collected by the FS method and the CNF method.

[0207] The present invention uses a plaque-forming assay to measure the DENV-2 titer in infected mosquito saliva. Materials: C6 / 36 cells (Cat#CRL-1660, ATCC) were cultured at 37°C in RPMI1640 medium (Cat#22400089, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Cat#ST30-3302, PAN) and 1% antibiotic-antimycotic (Cat#15240-062, Invitrogen). Vero cells (Cat#CCL81, ATCC) were grown in DMEM medium (Cat#C11995500BT, Gibco) supplemented with 10% FBS and 1% antibiotic-antimycotic. Dengue virus type 2 (DENV-2, New Guinea C strain, AF038403.1) was cultured in C6 / 36 cells using VP-SFM serum-free medium (Cat#11681-020, Gibco) and titered on Vero cells using a plaque-forming assay.

[0208] The specific detection method is as follows: Vero cells were plated in 24-well plates (2×10 5Cells were plated at 400 μL / well (200 μL / well) and incubated overnight. Cells were inoculated with serial dilutions of mosquito saliva (200 μL / well in DMEM containing 2% FBS) at 37°C for 1 hour, then washed with PBS and covered with medium. The plates were incubated at 37°C for 4-5 days. The covering medium was carefully removed, and the cells were fixed with 4% paraformaldehyde at 25°C for 30 minutes. The fixed cells were stained with 0.1% crystal violet (Cat#C6158, Sigma) in 10% ethanol (Cat#E7023, Sigma) at 25°C for 20 minutes to visualize plaques.

[0209] The results showed that the virus titer of mosquito saliva collected by FS method was 3.46PFU / saliva sample, while the virus titer of mosquito saliva collected by CNF method was 28.48PFU / saliva sample ( Figure 3 D), indicating that the CNF method can better maintain the infectious activity of the virus in mosquito saliva than the FS method. The reason may be that the CNF method does not use immersion oil and the mosquito saliva is collected under physiological conditions.

[0210] In summary, compared with the FS method, the CNF method can better preserve the biological activity of mosquito salivary proteins and the infectious activity of viruses in mosquito saliva.

[0211] Test Example 5: Overall comparison of the two methods

[0212] Table 1 summarizes the advantages of the CNF method over the FS method in terms of ease of operation, time savings, and collection efficiency. As shown in Table 1, compared to the FS method, the CNF method allows for the reuse of mosquitoes, is convenient to operate, saves time, and collects salivary proteins and viruses with far greater biological activity than the FS method. The FS method is time-consuming and prone to mosquito mortality, oil leakage, and residue during saliva collection, resulting in low mosquito salivary protein collection efficiency. Furthermore, the removal of wings and legs can cause mosquito mortality, preventing the mosquito from being fully recovered. Comparison of the two methods reveals that the CNF method is simple to operate, collects significantly more mosquito salivary proteins in a shorter period of time, and does not harm the mosquitoes, supporting the dynamic collection of mosquito salivary proteins and their reuse.

[0213] Table 1 Comparison of CNF method with FS method

[0214]

[0215] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0216] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for collecting proteins and / or viruses from mosquito saliva, characterized in that: The method comprises: a step of collecting saliva, wherein the mosquitoes whose saliva is to be collected for protein and / or virus are sucked on mosquito liquid food in a collecting component and secrete saliva into the collecting component, thereby obtaining mosquito liquid food containing saliva in the collecting component; a step of separating saliva, wherein the mosquito liquid food containing saliva in the collecting part is separated from the collecting part by centrifugation to obtain a first filtrate; a washing step, wherein the collecting component after centrifugation in the saliva separation step is soaked in a cleaning solution, and then the remaining saliva in the collecting component is separated from the collecting component by squeezing and / or centrifugation to obtain a second filtrate; The step of collecting proteins and / or viruses comprises mixing the first filtrate and the second filtrate, and concentrating the mixture to obtain proteins and / or viruses in mosquito saliva.

2. The method according to claim 1, characterized in that The cleaning solution comprises a solution composed of a weak acid and its conjugate acid salt. Preferably, the cleaning solution comprises at least one of phosphate buffer, acetate buffer and carbonate buffer. More preferably, the cleaning solution is phosphate buffer.

3. The method according to claim 1 or 2, characterized in that In the step of separating saliva and the step of washing, the centrifugation is performed at a temperature not higher than 10°C. Preferably, the centrifugation is performed at a temperature of 2-6°C.

4. The method according to any one of claims 1 to 3, characterized in that Before the step of collecting saliva, the mosquitoes whose saliva proteins and / or viruses are to be collected are fasted. Preferably, the fasting treatment time is within 72 hours.

5. The method according to any one of claims 1 to 4, characterized in that: The mosquito liquid food includes a sugar compound solution, Preferably, the mosquito liquid food comprises at least one of glucose solution, maltose solution, sucrose solution and lactose solution. More preferably, the carbohydrate solution comprises a sucrose solution.

6. The method according to claim 5, characterized in that The concentration of the sucrose solution is 5-20 (w / v)%. Preferably, the concentration of the sucrose solution is 8-15 (w / v)%.

7. The method according to any one of claims 1 to 6, characterized in that: In the step of collecting saliva, the mosquitoes whose saliva is to be collected for protein and / or virus feed on the mosquito liquid food in the collecting component under light-proof conditions.

8. The method according to any one of claims 1 to 7, characterized in that: In the saliva collecting step, the mosquitoes whose saliva is to be collected for protein and / or virus feed on the mosquito liquid food in the collecting component for 1 to 3 hours in a light-proof condition.

9. The method according to any one of claims 1 to 8, characterized in that In the step of collecting saliva, the collecting member is made of a material that is water-absorbent and does not contain biological protein components. Preferably, the collecting component is a sponge, More preferably, the collecting member is a sponge composed of at least one of polyurethane, polyvinyl alcohol or polyglycolic acid.

10. The method according to any one of claims 1 to 9, characterized in that: In the step of collecting proteins and / or viruses, the concentration is performed using an ultrafiltration centrifuge tube.

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