CN110713968A - Method for rapidly obtaining large amount of purified plasmodium sporozoites - Google Patents

Abstract

The invention relates to a method for rapidly obtaining a large amount of purified plasmodium sporozoites, which comprises the steps of firstly collecting anopheles mosquitoes infected with malaria, replacing the traditional homogenization treatment method with a fine dissection separation method after anesthesia and disinfection to obtain salivary gland sporozoites, and then replacing a traditional cellulose column with an aseptic filter membrane for purification treatment to obtain the purified plasmodium sporozoites. The operation is simple and reversible, and compared with the traditional method, the method has the advantages of short separation time, high sporozoite yield, high activity and the like.

Description

Method for rapidly obtaining large amount of purified plasmodium sporozoites

Technical Field

The invention belongs to the technical field of malaria research, and relates to a method for quickly obtaining a large amount of purified plasmodium sporozoites.

Background

Malaria is one of three major infectious diseases that seriously jeopardize public health, with about 40 million deaths worldwide each year. Anopheles mosquito is the vector of malaria, and when a person bites or bites to suck blood, sporozoites in the salivary glands then enter the person together to open up the malaria infection. Since sporozoites are the source of infection, various studies around sporozoites are the focus and emphasis in the malaria field, such as the migration pathway of sporozoites in the human body, obligate parasitization of sporozoites on hepatocytes, attenuated sporozoite vaccines, and the like. However, regardless of the study, it is first necessary to isolate sporozoites. The present invention aims at establishing a method for quickly obtaining a large amount of purified plasmodium sporozoites.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for rapidly obtaining a large amount of purified plasmodium sporozoites, which can be used in subsequent downstream experiments.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for quickly obtaining a large amount of purified plasmodium sporozoites comprises the steps of collecting anopheles infected with malaria, dissecting and stripping salivary glands of the anopheles under a microscope after anesthesia and immersion, releasing and collecting plasmodium sporozoites in the salivary glands through mechanical extrusion, and then purifying by using a sterile filter membrane to obtain the purified plasmodium sporozoites. Preferably, the plasmodium sporozoites are sporozoites of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale, plasmodium burgeri, plasmodium falciparum, or plasmodium yoelii.

Preferably, the anesthesia method is ether anesthesia, the immersion cleaning method comprises the steps of soaking and sterilizing 75% ethanol water solution by volume percentage, then soaking and cleaning with PBS, the microscope is a special dissecting microscope, and the tool for dissecting and stripping salivary glands is a medical sterile disposable 1ml syringe.

Further preferably, the anesthesia time is 2-3 minutes, and the specific method of immersion cleaning comprises the following steps: soaking with ethanol water solution for 1 min, removing liquid, adding PBS, soaking for 3 min, washing with PBS for 3 times, and placing anopheles on ice for dissection.

Preferably, the specific method of dissection is as follows:

(1) separating the head and the chest of the anopheles;

(2) gently squeezing anopheline thorax to slowly release salivary gland;

(3) separating out complete salivary gland, transferring to a sterile glass slide, and dripping liquid culture medium to prevent drying of salivary gland and keep vitality;

(4) covering the material on the sterile glass slide with a sterile cover glass, and collecting by squeezing the sterile cover glass to break down salivary glands to release plasmodium sporozoites.

Further preferably, the specific method of step (1) is: holding sterile syringes with two hands simultaneously, fixing anopheles chest with one syringe needle, then cutting off anopheles head with the knife edge of the other syringe needle to separate it from chest, paying attention to the gentle action of the process, not destroying anopheles chest, and cutting off rapidly without leading out anopheles thoracoabdominal viscera.

Further preferably, the specific method of step (2) is: fixing the abdomen of the anopheles mosquito by using one syringe needle, slightly pressing the chest of the anopheles mosquito by using the other syringe needle which is at an angle of about 45 degrees with the chest of the anopheles mosquito to slowly release salivary glands, and paying attention to the fact that the force is not too heavy when the pressing is carried out, otherwise, damaging the chest of the anopheles mosquito to avoid obtaining the salivary glands.

Further preferably, the specific method of step (3) is: the released salivary glands are often adhered to other tissues, intact salivary glands are carefully separated with a syringe needle, taking care that the salivary glands cannot be punctured at this time, otherwise the sporozoites will spill, carefully picked up in the center of a sterile clean slide, and simultaneously dropped with 200 μ L of ice-chilled 1640 medium to prevent the salivary glands from drying and remaining viable.

Further preferably, the specific method of step (4) is: the liquid edge on the slide was quickly lowered and covered with a sterile coverslip after contacting it with an angle of 45 ° and then the coverslip was squeezed with sterile ophthalmic forceps to disrupt the salivary glands to release sporozoites, the liquid was carefully aspirated into a sterile EP tube, then the slide was washed 3 times with 1640 medium, the liquid was collected into the EP tube and placed on ice to maintain viability.

Preferably, the pore size of the sterile filter membrane is 75 μm (200 mesh), and the filter membrane is commercially available and sterilized by high pressure steam sterilization or ultraviolet irradiation.

Further preferably, the sterile filter membrane is a nylon microporous filter membrane.

Preferably, the specific method of purification treatment is as follows:

(A) preparing 12 sterile filter membranes;

(B) adding 500 μ L1640 culture medium into the EP tube for collecting sporozoites, repeatedly blowing and beating with 1ml pipette for 30 times, fully breaking salivary gland again, and releasing more sporozoites;

(C) preparing 7 clean 1.5ml EP tubes, and placing the tubes on ice for precooling for 5 minutes; placing a filter membrane at the orifice of one EP tube, tightly holding the two sides with two hands to tighten the filter membrane, carefully sucking the sporozoite-containing liquid by a pipettor to enable the sporozoite-containing liquid to pass through the filter membrane, and repeating the step for 2 times after the process is finished; then, two filter membranes with 200 meshes are overlapped on the other clean EP pipe orifice, and the filtration is carried out for 3 times by the same method; and finally, overlapping three 200-mesh filter membranes on the other clean EP pipe orifice, and filtering for 1 time by the same method.

It is further preferred that step (C) is performed on ice throughout to maintain sporozoite viability.

The invention has the beneficial effects that:

according to the invention, anopheles mosquitoes infected with malaria are collected, after anesthesia and disinfection, a fine dissection method is used for replacing a traditional homogenization treatment method to obtain salivary gland sporozoites, and then a sterile filter membrane is used for replacing a traditional cellulose column for purification treatment, so that purified plasmodium sporozoites are obtained. The operation is simple and reversible, and compared with the traditional method, the method has the advantages of short separation time, high sporozoite yield, high activity and the like. The specific analysis is as follows:

1. the time is short: according to the traditional cellulose column separation method, at least 13 hours are needed from the preparation of a separation column (cellulose DE52 needs to be swelled in advance and usually needs to be soaked overnight) to the separation end, if the separation column configuration is not ideal, the column passing time of sporozoites is greatly increased, the steps are complicated, time and labor are wasted, special materials are not needed in the method, the whole separation process can be completed within about 1 hour, the experiment time is greatly shortened, and the efficiency is improved.

2. The yield is high: the traditional cellulose column separation method has the advantages that the sporozoite yield is low and unstable (14-50%), the number of steps is large, so that interference experiment factors are more, the sporozoite is lost a lot in the separation process, and if the experiment requirement is large, the sporozoite yield cannot be met, but the method has the sporozoite yield stabilized at more than 80%, and a large amount of purified sporozoites can be obtained through one-time separation.

3. The activity is high: the sporozoites obtained by the traditional cellulose column separation method have obviously reduced capability of invading the liver cells compared with the sporozoites obtained by the method because of too long separation time, which shows that the activity of the sporozoites is seriously influenced, while the method has little influence on the activity of the sporozoites and has no influence on the capability of invading the liver cells because of short separation time.

4. The operation is reversible: the traditional cellulose column separation method has complicated steps, and if sporozoites are added into cellulose with wrong preparation, the sporozoites cannot be obtained, and the operation is irreversible. The method is simple and reversible, and sporozoites cannot be recovered even if the nylon membrane is placed wrongly.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a graph comparing sporozoite separation times for two methods.

FIG. 2a is a graph comparing sporozoite yields for two methods; FIG. 2b is a comparison of sporozoite yields from the two methods using a light microscope.

FIG. 3 is a graph comparing sporozoite viability for the two methods.

FIG. 4 is a graph comparing the purity of sporozoites obtained by the two methods.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The present invention relates to a PBS having a pH of 7.4.

The method of the present invention is illustrated BY isolating sporozoites of Plasmodium yoelii BY265-GFP insect strain (with GFP green fluorescence), and it should be noted that the present invention is not limited to this sporozoite.

Example (b):

(1) separating plasmodium sporozoites;

the first step is as follows: collecting 100 infectious Anopheles sinensis (Hor strain which is bred and raised in a long-term in a basic medical institute of military and medical university, basic medical institute of the liberation army and military university of China), quickly and briefly anaesthetizing for 2-3 minutes, and transferring to a 50ml EP tube;

the second step is that: adding 30ml of 75% sterilized alcohol by volume percentage into an EP tube, soaking the infective anopheles sinensis for 1 minute, removing liquid, adding PBS (phosphate buffer solution) for soaking for 3 minutes, and repeatedly washing for 3 times;

the third step: preparing a clean glass slide, dripping 200 μ l 1640 culture medium into the center of the clean glass slide, and placing the glass slide on ice for later use;

the fourth step: dissecting infectious anopheles microscopically under a dissecting microscope, wherein the whole separation process needs to be carried out under the dissecting microscope. The first step, separating the head and the chest of the anopheles mosquito, the method is as follows: holding sterile syringes with two hands simultaneously, fixing the chest of the anopheles by using one syringe needle, and then cutting off the head of the anopheles by using the blade surface of the other syringe needle to separate the head of the anopheles from the chest, paying attention to the fact that the action of the process is as gentle as possible, the chest of the anopheles cannot be damaged, and the cutting process needs to be rapidly broken, so that the visceral organs of the chest and abdomen of the anopheles cannot be drawn; fixing the abdomen of the anopheline mosquito by using one syringe needle, slightly pressing the chest of the anopheline mosquito by using the other syringe needle which forms an angle of about 45 degrees with the chest of the anopheline mosquito, so that the anopheline mosquito slowly releases salivary glands, and paying attention to the fact that the force is not too heavy during pressing, otherwise, damaging the chest of the anopheline mosquito and failing to obtain the salivary glands; third, the released salivary gland is usually adhered with other tissues, the whole salivary gland is carefully separated by a syringe needle, the salivary gland cannot be punctured at the moment, otherwise, sporozoites overflow, the sporozoites are carefully picked to the center of a sterile clean glass slide, and 200 mu L of ice-precooled 1640 culture medium is dripped to prevent the salivary gland from being dried and keep vitality. Fourth, after all dissections were completed, a sterile coverslip was placed in contact with the edge of the liquid on the slide at a 45 degree angle and quickly laid down and covered, then the coverslip was squeezed with sterile ophthalmic forceps to disrupt the salivary glands and release the sporozoites, carefully aspirating the liquid into a sterile EP tube, then washing the slide 3 times with 1640 medium, collecting the liquid into the EP tube and placing on ice to maintain viability.

(2) Purifying plasmodium sporozoites;

the first step is as follows: preparing 12 sterile 200-mesh (75 μm) filter membranes;

the second step is that: adding a proper amount of 1640 culture medium into the EP tube for collecting the sporozoites in the previous step, repeatedly blowing and beating for 30 times by using a 1ml pipette gun, fully breaking salivary glands again, and releasing more sporozoites;

the third step: prepare 7 clean 1.5ml EP tubes, place a 200 mesh filter at one of the tube ports, hold both sides tightly with both hands to tighten it, pipette carefully aspirate the sporozoite-containing liquid through the nylon membrane, and repeat this step 2 times. Then, two filter membranes with 200 meshes are overlapped on the other clean EP pipe orifice, and the filtration is carried out for 3 times by the same method; and finally, overlapping three 200-mesh filter membranes on the other clean EP pipe orifice, filtering for 1 time in the same way, and placing the EP pipe on ice for waiting for counting.

(3) Counting plasmodium sporozoites;

the first step is as follows: the sporozoites obtained in the step (2) are mixed evenly by soft reversal, 10 mul of stock solution is absorbed to dilute 100 times, and then the diluted stock solution is added into a cell counting plate;

the second step is that: placing the cell counting plate under a microscope for counting, wherein the total number of sporozoites in four large lattices at four corners of the counting plate is counted, divided by 4, multiplied by dilution times and multiplied by 10⁴The density of the sporozoites in the stock solution is obtained, and finally the density is multiplied by the volume of the stock solution to obtain the total quantity of the sporozoites.

(4) Identifying the activity of plasmodium sporozoites;

the first step is as follows: reviving the hepatocyte Hepa1-6 frozen by liquid nitrogen, washing for 3 times by PBS, seeding in a cell culture bottle, adding 5ml of 1640 culture medium containing 10% (volume ratio) fetal calf serum, changing the culture medium the next day, and performing subculture in bottles when the cell fusion degree reaches about 90%;

the second step is that: mixing sporozoites obtained in the step (2) with hepatocyte Hepa1-6 in a quantitative ratio of 1: 2, CO at 37 ℃₂Placing the culture box for 42 hours;

the third step: collecting cultured cells, and quantitatively detecting sporozoites invading the hepatocytes by PCR (polymerase chain reaction), wherein the method is a gold standard for detecting the activity of the sporozoites and comprises the steps of extracting total RNA (ribonucleic acid) of the cells by TRIzol; after DNase I treatment, PrimeScript was used^TMRTreagen Kit with gDNA Eraser (Takara) reverse transcribes RNA to cDNAAs a template, a TaqMan RT-PCR reaction system was constructed, in which 20. mu.L of the system was prepared, 1. mu.L of cDNA, 1. mu.L of each of the upstream and downstream primers (upstream sequence: 5'-GGTTTTATAATTGGAATGATGGGAAT-3'; downstream sequence: 5'-ACGCTATTGGAGCTGGAATTACC-3'), 0.5. mu.L of the probe (FAM-AACCTTCCCAAAAAT-MG), 10. mu.L of Premix Ex Taq (2X), and H₂O6.5. mu.L. Amplification conditions: 2min at 95 ℃; at 95 ℃ for 15s and 60 ℃ for 20s, and 40 cycles; the relative 18sRNA content of each sample was determined from the 18srRNA/GAPDH ratio.

(5) Purity identification of plasmodium sporozoites

The first step is as follows: reviving the hepatocyte Hepa1-6 (purchased from American type culture Collection ATCC, with the product number of ATCCCRL-1830) frozen by liquid nitrogen, washing with PBS for 3 times, inoculating into a cell culture bottle, adding 1640 culture medium containing 10% (volume ratio) fetal calf serum 5ml, changing the culture medium the next day, and performing subculture when the cell fusion degree reaches about 90%;

the second step is that: mixing sporozoites obtained in the step (2) with hepatocyte Hepa1-6 in a quantitative ratio of 1: 2, placing in CO at 37 DEG C₂In the incubator, the purity of sporozoites is measured by observing the time during which the bacteria appear during the cell culture process.

The sporozoite separation time comparison (table 1, fig. 1), sporozoite yield comparison (fig. 2a, fig. 2b), sporozoite viability comparison (fig. 3), sporozoite purity comparison (fig. 4) was performed with the conventional method (cellulose column DE 52). From the results, it can be seen that the sporozoites obtained by the method of the present invention have the same purity as the conventional method, but the method of the present invention greatly shortens the separation time, improves the sporozoite yield, maintains the sporozoite vitality, and has reversible operation steps.

TABLE 1 comparison of sporozoite isolation times

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (7)

1. A method for quickly obtaining a large amount of purified plasmodium sporozoites is characterized by firstly collecting anopheles infected with malaria, dissecting and stripping salivary glands of the anopheles under a microscope after anesthesia and immersion, releasing and collecting plasmodium sporozoites in the salivary glands through mechanical extrusion, and then purifying by using a sterile filter membrane to obtain the purified plasmodium sporozoites.

2. The method of claim 1, wherein the plasmodium sporozoites are sporozoites of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale, plasmodium burgeri, plasmodium charnei, or plasmodium yoelii.

3. The method of claim 1, wherein the anesthesia method is ether anesthesia, the immersion method comprises immersion sterilization with 75% ethanol aqueous solution by volume percentage, followed by immersion and washing with PBS, the microscope is a special dissecting microscope, and the tool for dissecting and dissecting the salivary glands is a medical sterile disposable 1ml syringe.

4. The method according to claim 3, wherein the anesthesia time is 2-3 minutes, and the specific method of the immersion cleaning is as follows: soaking with ethanol water solution for 1 min, removing liquid, adding PBS, soaking for 3 min, washing with PBS for 3 times, and placing anopheles on ice for dissection.

5. The method of claim 1, wherein the specific method of dissection is:

(1) separating the head and the chest of the anopheles;

(2) gently squeezing anopheline thorax to slowly release salivary gland;

(3) separating out complete salivary gland, transferring to a sterile glass slide, and dripping liquid culture medium to prevent drying of salivary gland and keep vitality;

(4) covering the material on the sterile glass slide with a sterile cover glass, and collecting by squeezing the sterile cover glass to break down salivary glands to release plasmodium sporozoites.

6. The method of claim 1, wherein the pore size of the sterile filter is 75 μm.

7. The method according to claim 1, wherein the purification treatment is carried out by the following method:

(A) preparing 12 sterile filter membranes;

(B) adding 500 μ L1640 culture medium into the EP tube for collecting sporozoites, repeatedly blowing and beating with 1ml pipette for 30 times, fully breaking salivary gland again, and releasing more sporozoites;

(C) preparing 7 clean 1.5ml EP tubes, and placing the tubes on ice for precooling for 5 minutes; placing a filter membrane at the orifice of one EP tube, tightly holding the two sides with two hands to tighten the filter membrane, carefully sucking the sporozoite-containing liquid by a pipettor to enable the sporozoite-containing liquid to pass through the filter membrane, and repeating the step for 2 times after the process is finished; then, two filter membranes with 200 meshes are overlapped on the other clean EP pipe orifice, and the filtration is carried out for 3 times by the same method; and finally, overlapping three 200-mesh filter membranes on the other clean EP pipe orifice, and filtering for 1 time by the same method.

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