CN116731160A

CN116731160A - Recombinant protein vaccine for resisting long horn blood ticks and preparation method thereof

Info

Publication number: CN116731160A
Application number: CN202310705045.8A
Authority: CN
Inventors: 孙亚丽; 李积旭; 康明; 马鹤嘉
Original assignee: Qinghai University
Current assignee: Qinghai University
Priority date: 2023-06-14
Filing date: 2023-06-14
Publication date: 2023-09-12

Abstract

The invention discloses a recombinant protein vaccine for resisting long-horned blood ticks and a preparation method thereof, belonging to the technical field of biological medicines, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO.1, and the preparation method comprises recombinant vector construction, escherichia coli transformation, expression and purification. The recombinant protein vaccine is prepared from secreted proteins related to a coagulation mechanism of the long-angle blood ticks in the process of biting a host, can resist an anticoagulation mechanism of the long-angle blood ticks, can inhibit long-time blood suction of the long-angle blood ticks, avoids possibility of toxin dispersion compared with a traditional inactivated vaccine, reduces damage to animals, is more efficient, has a certain interference effect on biting and passaging of the long-angle blood ticks, enhances the protection of host animals compared with traditional chemical medicaments, reduces damage to animals, is more efficient, and has better application prospect and popularization value.

Description

Recombinant protein vaccine for resisting long horn blood ticks and preparation method thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant protein vaccine for resisting long horned blood ticks and a preparation method thereof.

Background

Hemicarx (Haemaphysalis Iongicornis), small tick, is yellow brown. There are no eyes and there are stacks of rims. The long-angle blood ticks can be parasitic to various hosts including human beings, domestic animals and wild animals, the hosts generally have no pain and itching feeling when biting, are difficult to perceive, and the neurotoxin secreted in the blood sucking process can lead to host muscle paralysis and can cause paralysis when severe. Long angle blood ticks are used as important pathogen carriers, can cause rickettsia, human monocytic iriasis, canine burnt worm disease, theileriosis, fever with thrombocytopenia syndrome and the like, and bring great harm to human health and animal husbandry development. At present, chemical control is still an important way to control ticks, but long-term use causes environmental pollution and tick resistance, so vaccination has become one of the effective tick control strategies.

Existing 19I SP-based mRNA vaccines, which do not direct cells in the body to produce spike proteins to train the body to attack SARS-CoV-2, but rather direct cells to produce some of the proteins found in the saliva of the black leg tick (Ixodes scapularis), prevent tick-induced disease. In addition, an anti-tick vaccine Bm86 was developed based on the intestinal glycoprotein of Rhipicephalus microplus (Rhipicephalus microplus) as an immunoreactive antigen. Because the two vaccine proteins are derived from black leg ticks or rhipicephalus micropus, the control effect on the long-angle blood ticks is not ideal.

Therefore, there is a need to develop a recombinant protein vaccine against long horned tick and a method for preparing the same.

Disclosure of Invention

The invention aims to provide a recombinant protein vaccine for resisting long-horned blood ticks and a preparation method thereof, which are used for solving the following technical problems: the existing tick vaccine has an unsatisfactory control effect on the long-angle blood ticks.

The aim of the invention can be achieved by the following technical scheme:

a recombinant protein vaccine for resisting long horned tick comprises rHlHemaIin (recombinant Haemaphysalis Iongicornis Hemalin) recombinant protein;

the amino acid sequence of the rHlHemalin recombinant protein is shown as SEQ ID NO.1, and the specific sequence is as follows.

SEQ ID NO.1：

QRNGFCRLPAEPGICRAFMPRYYFDVEKGQCEQFIYGGCKGNENNFETLKECQDACGEPERASDFEKADFETGCKAAPETGLCKASFERWFFNAASGECEEFIYGGCGGNDNNYENKEECEFACKY (wherein x represents a stop codon).

A preparation method of a recombinant protein vaccine for resisting long horned blood ticks comprises the following steps:

step one, constructing a recombinant vector: PCR amplification is carried out by taking cDNA of the long horn blood ticks as a template to obtain a target fragment, and the target fragment is connected with pGEX-4T-1 vector to obtain recombinant plasmid pGEX-4T-1-kunitz;

step two, conversion: the recombinant plasmid pGEX-4T-1-kunitz is successfully transformed into escherichia coli BL21 (DE 3) and then is subjected to expansion culture, 10mL of bacterial liquid after expansion culture and 1mL of ampicillin with the concentration of 50 mug/mL are added into a 1LLB liquid culture medium, and the bacterial liquid is obtained after expansion culture for 3 hours under the shaking condition of 37 ℃ and 160 rpm;

step three, expression: adding IPTG (isopropyl-beta-D-thiogalactoside) with the molar concentration of 1M and DTT (dithiothreitol) with the molar concentration of 1M into the expanded bacterial liquid obtained in the step two to ensure that the concentrations of the IPTG and the DTT are 0.2mM, and culturing for 20 hours under the shaking condition of 25 ℃ and 120rpm to obtain a protein expression liquid;

step four, purifying: centrifuging the protein expression liquid with a centrifuge at 6000rpm for 10min, removing supernatant, washing the collected thalli with PBS buffer for 3 times, performing ultrasonic crushing, centrifuging at 6000rpm for 10min, sucking the supernatant, placing the supernatant into a clean centrifuge tube, and centrifuging at 12000rpm for 10min to obtain supernatant containing rHlHemalin soluble recombinant protein and precipitate containing inclusion body protein; purifying the supernatant containing rHlHemalin soluble recombinant protein to obtain rHlHemalin recombinant protein, i.e. a recombinant protein vaccine against long horned tick.

As a further aspect of the present invention, the PCR amplified primers include an upstream primer F-rHltihECORI and a downstream primer R-rHltihXhoI; the nucleotide sequence of the upstream primer F-rHltihECORI is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer R-rHltihXhoI is shown as SEQ ID NO.3, and the specific sequence is as follows.

SEQ ID NO.2：

gcgaattctcagcgaaatggcttctgtcg

SEQ ID NO.3：

gcctcgagtcaatatttgcaggcgaactc。

As a further scheme of the invention, the nucleotide sequence of the target fragment is shown as SEQ ID NO.4, and the specific sequence is as follows.

SEQ ID NO.4：

cagcgaaatggcttctgtcggcttccggcggagcctgggatctgccgggcgtttatgccccgttattactttgacgtcgagaagggacagtgcgagcaattcatctacggaggatgcaaaggaaacgagaacaacttcgagactctaaaagaatgccaggacgcctgtggtgaacccgagagggcgagcgacttcgagaaggccgactttgaaaccggctgcaaggcagctcctgaaaccggactttgcaaggctagctttgagcgctggttcttcaatgccgcctcgggcgagtgcgaggagtttatttatggcggttgcggtggcaacgacaacaactatgagaacaaggaggaatgcgagttcgcctgcaaatattga

As a further aspect of the present invention, the specific steps of purifying the supernatant containing rHlHemaIin soluble recombinant protein in the fourth step are as follows:

adding 1mL of glutathione affinity filler (Glutathione Sepharose TM B) into a chromatographic column, adding PBS buffer to balance the filler, adding supernatant containing rHlHemalin soluble recombinant protein after balancing for 4-6 times, washing for 4-5 times with PBS buffer, washing off foreign proteins, adding eluent to elute target proteins, adding target proteins into a dialysis bag, and dialyzing at 4 ℃ for 16-20h to obtain purified rHlHemalin soluble recombinant protein. The dialyzed proteins were aspirated into centrifuge tubes and stored at-80℃with sealing membranes.

The invention has the beneficial effects that:

the recombinant protein vaccine is prepared from secreted proteins related to a coagulation mechanism of the long-angle blood ticks in the process of biting a host, can resist an anticoagulation mechanism of the long-angle blood ticks, can inhibit long-time blood suction of the long-angle blood ticks, and compared with a traditional inactivated vaccine, the recombinant protein vaccine has the advantages of avoiding possibility of toxin dispersion, reducing harm to animals and being more efficient.

The recombinant protein vaccine for resisting the long-angle blood ticks has a certain interference effect on biting and passaging of the long-angle blood ticks, enhances the protection of host animals compared with the traditional chemical medicaments, reduces the damage to the animals, is more efficient, and has better application prospect and popularization value. Is beneficial to promoting the development of animal husbandry economy in the area of long-angle blood tick distribution, reducing the pollution of local water and soil resources, and laying a foundation for the development of related vaccines of other ticks while contributing to the prevention and treatment of long-angle blood tick-borne diseases.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 is a SDS-PAGE diagram of rHlHemalin recombinant protein of example 1, GST tag protein of comparative example 1 according to the present invention;

FIG. 2 is a graph showing the levels of antibodies before and after immunization of rabbits in the rHlHemalin immunized group and GST control group of the present invention;

FIG. 3 is a graph showing the physiological index observations of Haemophilus parasuis in the rHlHemalin immune group, GST control group and PBS control group of the present invention;

FIG. 4 is a statistical plot of anti-long blood tick infection of rHlHemalin immunized group, GST control group and PBS control group of the invention.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

step one, constructing a recombinant vector: PCR amplification is carried out by taking cDNA of the haemaphysalis longifolia as a template to obtain a target fragment, and after sequencing is correct by Beijing engine biotechnology limited company, the target fragment is connected with pGEX-4T-1 vector to obtain recombinant plasmid pGEX-4T-1-kunitz;

the PCR amplification system is shown in Table 1, wherein the nucleotide sequence of the upstream primer F-rHltihECORI is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer R-rHltihXhoI is shown as SEQ ID NO.3, and the specific sequences are as follows.

SEQ ID NO.2：

gcgaattctcagcgaaatggcttctgtcg

SEQ ID NO.3：

gcctcgagtcaatatttgcaggcgaactc。

TABLE 1

System component	System addition amount
		10 XThermopol buffer	5μL
10mM dNTPs	1μL
		10 mu M upstream primer F-rHltihECORI	1μL
10 mu M downstream primer R-rHltihXhoI	1μL
		cDNA of Haemophilus longifolius	1.5μL
TaqDNA polymerase	0.25μL
		RNAase-Free Water	40.25μL

The PCR reaction procedure is shown in Table 2:

TABLE 2

The nucleotide sequence of the target fragment with correct sequencing is shown as SEQ ID NO.4, and the specific sequence is as follows.

SEQ ID NO.4：

Step two, conversion: the recombinant plasmid pGEX-4T-1-kunitz is successfully transformed into escherichia coli BL21 (DE 3) and then is subjected to expansion culture, 10mL of bacterial liquid after expansion culture and 1mL of ampicillin with the concentration of 50 mug/mL are added into 1L of LB liquid medium, and the bacterial liquid is obtained after expansion culture for 3 hours under the shaking condition of 37 ℃ and 160 rpm;

step three, expression: adding IPTG with the molar concentration of 1M and DTT with the molar concentration of 1M into the expanded bacterial liquid obtained in the step two to ensure that the concentrations of the IPTG and the DTT are 0.2mM, and culturing for 20 hours at 25 ℃ under the condition of shaking at 120rpm to obtain a protein expression liquid;

step four, purifying: centrifuging the protein expression liquid with a centrifuge at 6000rpm for 10min, removing supernatant, washing the collected thalli with PBS buffer for 3 times, performing ultrasonic crushing, centrifuging at 6000rpm for 10min, sucking the supernatant, placing the supernatant into a clean centrifuge tube, and centrifuging at 12000rpm for 10min to obtain supernatant containing rHlHemalin soluble recombinant protein and precipitate containing inclusion body protein;

adding 1mL of glutathione affinity filler into a chromatographic column, adding PBS buffer to balance the filler, balancing for 5 times, adding supernatant containing rHlHemalin soluble recombinant protein, washing for 4 times by using the PBS buffer, washing off the impurity protein, adding eluent to elute the target protein, adding the target protein into a dialysis bag, and dialyzing at 4 ℃ for 16 hours to obtain purified rHlHemalin soluble recombinant protein, wherein the amino acid sequence is shown as SEQ ID NO.1, and the specific sequence is as follows.

SEQ ID NO.1：

QRNGFCRLPAEPGICRAFMPRYYFDVEKGQCEQFIYGGCKGNENNFETLKECQDACGEPERASDFEKADFETGCKAAPETGLCKASFERWFFNAASGECEEFIYGGCGGNDNNYENKEECEFACKY (wherein represents a stop codon)

The dialyzed proteins were aspirated into centrifuge tubes and stored at-80℃with sealing membranes.

Comparative example 1

Purification of GST tag protein comprising the steps of:

step 1: e.coli BL21 (DE 3) was transformed with pGEX-4T-1 empty vector and subjected to expansion culture, 10mL of pGEX-4T-1 empty vector transformed E.coli BL21 (DE 3) broth and 1mL of ampicillin having a concentration of 50. Mu.g/mL were added to 1LLB liquid medium, and cultured at 37℃and 160rpm shaking for 3 hours.

Step 2: IPTG and DTT were added to the LB liquid medium to a final concentration of 0.2mM, and cultured at 25℃for 20 hours under shaking at 120 rpm; the collected cells were centrifuged at 6000rpm for 10min, and the collected cells were washed with PBS buffer 5 times.

Step 3: ultrasonically crushing the washed thickness thalli, centrifuging for 10min under the condition of 6000rpm, sucking the supernatant, placing into a clean centrifuge tube, centrifuging for 10min under the condition of 12000rpm, and washing the precipitate with PBS buffer solution for 5 times; and (3) carrying out chromatography after re-suspending the precipitate, eluting the target protein and collecting the target protein to obtain the GST tag protein.

Performance testing

(1) SDS-PAGE analysis:

SDS-PAGE analysis is performed on the purified proteins of example 1 and comparative example 1, and the results are shown in FIG. 1, wherein the 1-2 hole sites are the recombinant protein vaccine prepared in example 1, and the 3-4 hole sites are GST tag proteins purified in comparative example 1, so that the recombinant protein vaccine prepared in example 1 successfully expresses Hemalin protein;

(2) attack infection detection and long angle blood tick physiological index statistical analysis:

step (1), the New Zealand white rabbits were randomly divided into 3 groups of three, respectively labeled rHlHemalin immune group, GST control group and PBS control group, and immunized according to Table 3: the first immunization and the second immunization were separated by 14 days, and the second and third immunization were separated by 7 days.

TABLE 3 Table 3

And (2) respectively taking blood to collect serum after each immunization of the rHlHemalin immune group and the GST control group, detecting the antibody level, and detecting the collected serum sample by an enzyme-linked immunosorbent assay (ELISA) method to detect IgG antibodies generated by the recombinant protein vaccine in the rabbit serum. The specific method comprises the following steps:

s1, coating: protein was coated in 96-well plates at an optimal concentration of 0.25. Mu.g/ml with coating solution, and 100. Mu.L of coating solution was added to each well. Placing in a refrigerator at 4deg.C for 16 hr.

S2, washing: taking out the 96-well plate, throwing out the content, adding 200 mu L of washing liquid by a discharge gun, standing for 3min, discarding the washing liquid, and beating with absorbent paper.

S3, sealing: 100. Mu.L of blocking solution per well was added and incubated in an incubator at 37℃for 1h.

S4, washing: and (2) repeating the step (2), and if the refrigerator is unused within one week, sealing and storing the refrigerator in a refrigerator at the temperature of minus 20 ℃.

S5, the sample to be detected acts as follows: the samples to be examined were diluted (1:500 diluted with skim milk, 50. Mu.L per well transfer), added to a closed assay plate, and incubated at 37℃for 1 hour.

S6, washing: the washing was repeated 5 times.

S7, secondary antibody: diluted 1:4000 with skim milk, 50. Mu.L of IgG secondary antibody was added to each well and incubated at 37℃for 1h.

S8, washing: the washing was repeated 5 times.

S9, developing: 50 mu L of color development liquid is added to each well under the dark environment, and the mixture is incubated at the constant temperature of 37 ℃ for 1h. S10, reading: the 96-well plate was placed in an microplate reader and measured at a wavelength of 405 nm.

As a result, as shown in FIG. 2, after the first immunization, the antibody level started to rise, and the antibody level remained stable until the third immunization ended, with OD value around 1.5, significantly higher than that of the GST control group (p < 0.05). The antibody level of the GST control group is not statistically different from that of serum before and after immunization, and can be used for subsequent insect attack experiments.

Step (3), after the immune titer of 3 groups of rabbits reaches a certain level, an attack experiment can be carried out, and the long-angle blood ticks are fed on the rabbit ears, and the specific operation is as follows:

firstly, a white cotton cloth is sewn into a straight cylinder with two open ends, the size is that the double ears of a rabbit can be wrapped, and animal gel is used for fixing, so that long-angle blood ticks are prevented from climbing out. One end of a straight cylinder-shaped cotton cloth cylinder is fixed on rabbit ears by animal gel, and the other end of the straight cylinder-shaped cotton cloth cylinder is penetrated by cotton threads to form a contractible closed port;

the scarf is made of corrugated paper board, the scarf is sleeved on the head of a rabbit, the long-angle blood ticks are connected in, and the cotton cloth and the rabbit ears are fast entangled by taking medical adhesive tapes. 45 parthenogenetic strain long horn blood ticks are inoculated to each rabbit;

each physiological index of the long-angle blood ticks is observed every day, and indexes such as the number of the adhered long-angle blood ticks, the time of saturation, the time of spawning of the long-angle blood ticks and the time of finishing spawning are recorded on each rabbit (blood saturation period: time period from adhesion to saturation of the long-angle blood ticks; pre-spawning period: time period from saturation of the long-angle blood ticks to pre-spawning; spawning period: time period from start of spawning to finishing of spawning; adhesion rate = (number of adhered long-angle blood ticks/total number of inoculated) x 100%, and blood saturation rate = (number of saturated-angle blood ticks/total number of adhered) x 100%).

And (4) finishing the collected data of the long-angle blood ticks, and performing biological analysis on each physiological index by adopting a One-way analysis of variance (One-wayANOVA) method by adopting GraphpadPrism software.

Each rabbit was inoculated with 45 parthenogenetic strain long horn blood ticks, and its physiological index was observed daily. On day 2 after attack, the rHlHemalin immunized group had a portion of the long horned ticks unattached (see FIG. 3 a) and the control group had all adhered (see FIG. 3 bc). On day 5 after attack, the rHlHemalin immune group had a dry and flat status (see FIG. 3 d) and the control group had a distended status (see FIG. 3 ef). The long-angle blood ticks grow up to about ten times the original volume and naturally drop off (see fig. 3 ghi).

(3) The collected saturated blood ticks are weighed and recorded, placed in a constant temperature incubator at 25 ℃ to be incubated in a dark place with enough humidity, and physiological indexes such as spawning period, egg weight and the like are recorded (see fig. 4).

The saturation period of the rHlHemalin immune group and the PBS control group is 5-7 days, and the PBS control group is 6-8 days, so that the rHlHemalin immune group and the PBS control group have extremely significant difference (p < 0.001) in the saturation period of the long-angle blood ticks, which indicates that the rHlHemalin recombinant protein vaccine can influence the normal saturation period of the long-angle blood ticks. The long-angle blood ticks begin to end the saturation period at day 5-7, which is reduced compared to the normal saturation period. We counted the body weight of the full blood after each group of 45 long-angle blood ticks had absorbed blood, the full blood weight of the long-angle blood ticks of the rHlHemalin immunized group was 83-284 mg, the PBS control group was 90-277mg, and the long-angle blood ticks of the rHlHemalin immunized group had significant differences from the PBS control group (p < 0.001). The period of starting spawning by the long horned ticks of the rHlHemalin immunized group was 4-7 days, which was no different from the PBS control group for 4-8 days. The egg laying weight of the long-angle blood ticks saturated by the rHlHemalin immune group is 35-1599 mg, the PBS control group is 51-171mg, and the egg laying weight of the long-angle blood ticks in the rHlHemalin immune group is not different from that in the PBS control group. For the egg incubation period of the long angle blood ticks, the rHlHemalin immune group is 14-36 days, the PBS control group is 10-38 days, the rHlHemalin immune group has extremely significant difference (p < 0.001) from the PBS control group, the egg incubation period of the rHlHemalin immune group is prolonged compared with the PBS control group, even part of ticks of the immune group are not hatched and die directly, which indicates that the rHlHemalin recombinant protein vaccine influences the normal egg incubation period of the long angle blood ticks. Comparing the ratio of the weight of the eggs of the long-angle blood ticks to the weight of the eggs of the complete ticks in the rHlHemalin immunized group to the PBS group, it can be found that the oviposition of the experimental group and the control group also has a significant effect (p < 0.01), and the above data comprehensively indicate that the rHlHemalin recombinant protein vaccine can affect the oviposition of ticks.

In conclusion, the recombinant protein vaccine has better effect on preventing and treating the long horned ticks.

It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A recombinant protein vaccine for resisting the long horned tick is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.

2. The method for preparing a recombinant protein vaccine against haemagglutinin according to claim 1, comprising the steps of:

step one, constructing a recombinant vector: PCR amplification is carried out by taking cDNA of the long horn blood ticks as a template to obtain a target fragment, and the target fragment is connected with pGEX-4T-1 vector to obtain recombinant plasmid pGEX-4T-1-kuni tz;

step two, conversion: successfully transforming recombinant plasmid pGEX-4T-1-kuni tz into escherichia coli BL21, performing amplification culture, sucking 10mL of bacterial liquid after amplification culture and 1mL of ampicillin with the concentration of 50 mug/mL into 1L of LB liquid medium, and culturing for 3 hours at 37 ℃ and 160rpm oscillation to obtain the amplified bacterial liquid;

step four, purifying: centrifuging the protein expression liquid with a centrifuge at 6000rpm for 10min, removing supernatant, washing the collected thalli with PBS buffer for 3 times, performing ultrasonic crushing, centrifuging at 6000rpm for 10min, sucking the supernatant, placing the supernatant into a clean centrifuge tube, and centrifuging at 12000rpm for 10min to obtain supernatant containing rHlHemalin soluble recombinant protein and precipitate containing inclusion body protein; purifying the supernatant containing rHlHemalin soluble recombinant protein to obtain rHlHemaliin recombinant protein, i.e. a recombinant protein vaccine against long horned tick.

3. The method for preparing a recombinant protein vaccine against haemaphysalis longifolia according to claim 2, characterized in that the PCR amplified primers comprise an upstream primer F-rHltihEcoRI and a downstream primer R-rhltixhohi; the nucleotide sequence of the upstream primer F-rHltihECORI is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer R-rHltihXhoI is shown as SEQ ID NO. 3.

4. The method for preparing the recombinant protein vaccine against the long horned tick according to claim 2, wherein the nucleotide sequence of the target fragment is shown as SEQ ID NO. 4.

5. The method for preparing recombinant protein vaccine against long horned tick according to claim 2, wherein the specific steps of purifying the supernatant containing rHlHemalin soluble recombinant protein in step four are as follows:

adding 1mL of glutathione affinity filler into a chromatographic column, adding PBS buffer to balance the filler, adding supernatant containing rHlHemalin soluble recombinant protein after balancing for 4-6 times, washing for 4-5 times by using PBS buffer, adding eluent to elute target protein, adding the target protein into a dialysis bag, and dialyzing at 4 ℃ for 16-20 hours to obtain purified rHlHemalin soluble recombinant protein.