Human three-type parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof

文档序号：62661 发布日期：2021-10-01 浏览：35次中文

阅读说明：本技术 一种人三型副流感病毒冷适应温度敏感株及其应用 (Human three-type parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof ) 是由白慕群火文关文竹李雄雄王云瑾包红寇桂英韩平王名强于 2021-03-24 设计创作，主要内容包括：本发明提供了一种人三型副流感病毒冷适应温度敏感株及其应用,所述人三型副流感病毒冷适应温度敏感株为HPIV3CPW3以及包含人三型副流感病毒冷适应温度敏感株的组合物,该人三型副流感病毒株HPIV3CPW3作为三型副流感病毒减毒活疫苗候选毒株,具有剂次效应以及良好的安全性,而且对小鼠肺部HPIV-3野毒株的感染有显著性保护效力,具有冷适应温度敏感特性的人三型副流感减毒活疫苗将能够有效预防三型副流感病毒的感染,而且该人三型副流感病毒株HPIV3CPW3具有冷适应和温度敏感特性且在小鼠的上下呼吸道也具有温度敏感的特性,这对于在接种的哺乳动物体内激发保护性免疫应答而不产生严重症状具有重要意义,可用于制备在对象中引起保护性免疫应答的药物。(The invention provides a human three-type parainfluenza virus cold-adaptive temperature-sensitive strain and an application thereof, the human three-type parainfluenza virus cold-adaptive temperature-sensitive strain is HPIV3CPW3 and a composition containing the human three-type parainfluenza virus cold-adaptive temperature-sensitive strain, the human three-type parainfluenza virus strain HPIV3CPW3 is used as a candidate strain of a three-type parainfluenza virus attenuated live vaccine, has secondary effect and good safety, has significant protective effect on the infection of mouse lung HPIV-3 wild strain, the human three-type parainfluenza virus attenuated live vaccine with the cold-adaptive temperature-sensitive characteristic can effectively prevent the infection of the three-type parainfluenza virus, and the human three-type parainfluenza virus strain HPIV3CPW3 has the cold-adaptive and temperature-sensitive characteristics and the temperature-sensitive characteristics on the upper and lower respiratory tracts of mice, which has important significance for exciting protective immune response in vaccinated mammals without generating severe symptoms, can be used for preparing a medicament for eliciting a protective immune response in a subject.)

1. The human tri-parainfluenza virus cold-adaptation temperature-sensitive strain is characterized by being HPIV3CPW3 with the preservation number of CCTCC NO: V202113 and being preserved in China center for type culture collection with the preservation date of 2021 year, 1 month and 22 days.

2. A composition comprising the human parainfluenza virus trio cold-adapted temperature sensitive strain of claim 1.

3. The composition of claim 2, wherein: the composition further comprises a pharmaceutically acceptable carrier or adjuvant.

4. The composition of claim 2, wherein: the composition is a human parainfluenza virus III vaccine with cold-adaptation temperature sensitivity.

5. The composition of claim 4, wherein: the human three-type parainfluenza vaccine with the cold-adaptation temperature sensitivity characteristic is a HPIV-3 attenuated live vaccine.

6. A method for producing a human parainfluenza virus serotype three vaccine having cold-adapted and temperature-sensitive properties, comprising using the human parainfluenza virus serotype three temperature-sensitive strain according to claim 1.

7. Use of the human parainfluenza virus triple cold-adapted temperature sensitive strain of claim 1 or the composition of any one of claims 2 to 5 in the manufacture of a medicament for eliciting a protective immune response in a subject.

8. Use according to claim 7, characterized in that: the protective immune response protects the subject against a disease caused by human parainfluenza virus III.

Technical Field

The invention relates to a human parainfluenza virus III, in particular to a human parainfluenza virus III cold-adaptation temperature-sensitive strain and application thereof.

Background

Human parainfluenza virus III (HPIV-3) is the second main pathogen of respiratory syncytial virus and is easy to cause acute lower respiratory diseases such as severe bronchitis and pneumonia of infants. HPIV-3 infection is very common among hospitalized children with acute respiratory infections, outbreaks of HPIV-3 occur annually, mainly in both spring and summer, and epidemiological studies have shown that 80% of infants under 5 years old are infected with HPIV-3. In addition, HPIV-3 infection is also a significant cause of lower respiratory tract diseases and death in elderly chronic patients and immunocompromised adults, and the death rate caused by HPIV-3 infection varies from 3% to 47% among organ transplant and immunosuppressed patients, causing a great disease burden. Therefore, research on the development of HPIV-3 vaccines is necessary.

In the development of HPIV-3 vaccines, inactivated vaccines were abandoned due to their disease-enhancing effects after vaccination, and current vaccine development has focused primarily on live attenuated and subunit vaccines. Two attenuated live vaccines developed by American scientists, one based on cross-protection of heterologous related viruses, bovine parainfluenza virus type III (bPIV-3) is considered as a promising candidate vaccine, and the second-phase clinical results show that the bPIV-3 attenuated live vaccine has good safety and tolerance in infants, can induce a bPIV-3 serum antibody response and has cross-protection efficacy on HPIV-3 infection; another promising candidate vaccine strain is HPIV-3 attenuated strain cp45 with temperature sensitivity obtained by cold adaptation passage of HPIV-3 virus, and research shows that cp45 has stronger immunogenicity than bPIV-3, and second-phase clinical results show that cp45 has good safety and tolerance, and induces higher neutralizing antibody response and protective efficacy in serum HPIV-3 antibody negative infants. Animal experiments show that the recombinant chimeric protein has high immunogenicity, can induce neutralizing antibody response of animals and can protect HPIV-3 infection, but the research of the subunit vaccine is still in a laboratory stage at present.

Until now, no specific medicine and preventive vaccine for effectively treating HPIV-3 infection is available on the market, so that the development of HPIV-3 cold-adaptation temperature sensitive attenuated vaccine is of great significance.

Disclosure of Invention

The invention aims to solve the technical problem of the prior art and provides a human tri-parainfluenza virus cold-adapted temperature-sensitive strain and application thereof.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: the human tri-parainfluenza virus cold-adaptation temperature-sensitive strain is characterized by being HPIV3CPW3 with the preservation number of CCTCC NO: V202113 and being preserved in China center for type culture collection with the preservation date of 2021 year, 1 month and 22 days.

The invention also provides a composition containing the human parainfluenza virus III cold-adapted temperature-sensitive strain.

Further, the composition further comprises a pharmaceutically acceptable carrier or adjuvant.

Further, the composition is a human parainfluenza triple vaccine with cold-adapted temperature sensitive properties.

Further, the human parainfluenza virus III with the cold-adaptation temperature sensitivity characteristic is a HPIV-3 attenuated live vaccine.

The invention also provides a preparation method of the human three-type parainfluenza vaccine with cold adaptation and temperature sensitivity characteristics, which comprises the step of using the human three-type parainfluenza virus cold adaptation temperature sensitive strain.

The invention also provides the use of a human parainfluenza virus triple cold-adapted temperature sensitive strain or a composition comprising a human parainfluenza virus triple cold-adapted temperature sensitive strain as described above in the preparation of a medicament for eliciting a protective immune response in a subject.

Further, the protective immune response protects the subject against a disease caused by human parainfluenza virus type III.

Compared with the prior art, the invention has the following beneficial effects: the invention provides a human three-type parainfluenza virus cold-adaptation temperature-sensitive strain HPIV3CPW3, the human three-type parainfluenza virus strain HPIV3CPW3 is used as a candidate strain of a three-type parainfluenza virus attenuated live vaccine, and a nasal cavity is used for immunizing a mouse, so that the safety is good; can induce the obvious serum neutralizing antibody response of the mouse and has the effect of dose; and has significant protective efficacy on the infection of mouse lung HPIV-3 wild strain. The human three-type parainfluenza attenuated live vaccine with the cold adaptation temperature sensitivity characteristic can effectively prevent the infection of three-type parainfluenza virus, and the human three-type parainfluenza virus strain HPIV3CPW3 has the cold adaptation and temperature sensitivity characteristics and also has the temperature sensitivity characteristic on the upper respiratory tract and the lower respiratory tract of a mouse, and the technical characteristic has important significance for exciting protective immune response in the inoculated mammal without generating serious symptoms.

Drawings

FIG. 1 shows the results of detection of an HPIV-3 positive specimen in example 1 of the present invention;

FIG. 2 shows the positive results of the primers for detecting foreign factors in example 1 of the present invention, wherein 1 is rhinovirus, 2 is adenovirus, 3 is mycoplasma, 4 is respiratory syncytial virus, 5 is influenza A H1N1 virus, 6 is influenza A H2N3 virus, and 7 is influenza B virus;

FIG. 3 shows the passaging results at 25 ℃ for the HPIV3CPW3 strain of example 1 according to the invention;

FIG. 4 is an electron micrograph of HPIV3CPW3 virus after ultracentrifugation purification according to example 2 of the present invention;

FIG. 5 shows the Western-blotting identification of HPIV3CPW3 virus after ultracentrifugation purification in example 2 of the present invention;

FIG. 6 shows the lesions of the HPIV3CPW3 strain of example 5 of the invention in cell culture at specific temperatures;

FIG. 7 shows the results of temperature-sensitive detection of HPIV3CPW3 virus and its parental wild strain LZ1501W3 on WI-38 cells in example 5 of the present invention;

FIG. 8 shows the identification results of the cold-adapted temperature-sensitive characteristics of the HPIV3CPW3 virus and its parental wild strain LZ1501W3 in the upper and lower respiratory tracts of mice in example 5 of the present invention;

FIG. 9 is a graph of a mouse serum HPIV-3 neutralizing antibody plaque reduction assay after immunization with HPIV3CPW3 virus according to example 6 of the present invention; wherein, the plaque patterns of the serial diluted experimental group serum and the HPIV3LZ1728C19 virus after neutralization are arranged in 1-9 holes, and the plaque patterns of the negative control group serum after 20-fold dilution and the HPIV3LZ1728C19 virus after neutralization are arranged in 10-12 holes;

FIG. 10 shows the results of the proliferation of the upper and lower respiratory tracts of mice immunized with the HPIV3CPW3 virus 3 agent of example 7 of the present invention;

FIG. 11 shows the safety evaluation results of mice immunized with HPIV3CPW3 virus according to example 7 of the present invention;

FIG. 12 shows the results of the neutralizing antibody responses of mouse serum HPIV-3 after immunization with HPIV3CPW3 virus according to example 7 of the present invention;

FIG. 13 shows the results of the evaluation of the protective efficacy of the HPIV3CPW3 virus immunization of mice lung HPIV-3 infection according to example 7 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.

The invention provides a human three-type parainfluenza virus strain HPIV3CPW3 with cold-adaptation temperature sensitivity, which is preserved in China center for type culture Collection (CCTCC, Wuhan university No. 299 in eight roads in Wuhan city, Hubei province) with the preservation number of CCTCC NO: V202113 and the preservation date of 2021 year, 1 month and 22 days.

Example 1 screening of Cold-adapted temperature-sensitive strains of human parainfluenza virus III

1.1 screening of HPIV-3 Positive clinical specimens

Screening materials: lower respiratory secretion specimens of infants who are hospitalized with lower respiratory infection in women and children health care hospitals in Gansu province are frozen at minus 80 ℃;

the screening method comprises the following steps: melting the virus screening material at room temperature, and then performing vortex oscillation for 2min at 12000 rpm; after the oscillation is finished, absorbing the vortex liquid into a 1.5ml centrifuge tube, and centrifuging for 20min at 12000 rpm; sterilizing and filtering the centrifuged supernatant by using a needle filter (0.22 mu m), inoculating the filtrate into a 6-well plate in which WI-38 (human embryonic lung fibroblast) grows, and incubating the filtrate at 37 ℃ for 2 hours; the supernatant was aspirated, 3ml of DMEM medium containing 10ug/ml mycoplasma removing agent Plasmocin (Invivogen, ant-mpt) was added thereto, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 7 days, continuously carrying out 10 times of hole-to-hole passage, and carrying out RT-PCR detection; taking a culture hole with positive HPIV-3, and carrying out virus titer determination by using qRT-PCR; as a result, the titer of one specimen was found to reach 3.2X10⁷Copy/ml, this HPIV-3 positive virus specimen was designated LZ1501 specimen as in fig. 1 for subsequent plaque cloning.

1.2 plaque clone purification of HPIV-3 Positive cultures

Serial 10-fold dilution of LZ1501 positive culture supernatant from 10 serial subcultures was performed at 10-fold^-1To 10^-8Doubling; then, the cells were seeded into 6-well plates in which WI-38 cells were grown, 10^-3To 10^-81 well of each dilution was inoculated with 1ml of each well and 5% CO at 37 ℃ after completion of inoculation₂The cells were cultured in a cell culture chamber for 2 hours, the supernatant was discarded, 3ml of DMEM-prepared low melting point agarose (Sigma, A9045) was added to each well, and after solidification at room temperature, the mixture was inverted at 37 ℃ and 5% CO₂Culturing for 4 days in a cell culture box; adding 1ml of 1% low melting point agarose prepared with DMEM containing neutral red, solidifying at room temperature, inverting at 37 deg.C and 5% CO₂Culturing for 24 hours in a cell culture box; selecting plaque from high dilution well of WI-38 cells, inoculating on the same cell in 24-well plate, and culturing at 37 deg.C and 5% CO₂Culturing for 7 days in a cell culture box; detecting the HPIV-3 positive clone culture by qRT-PCR, then picking 12 HPIV-3 positive initial clones, continuously cloning each HPIV-3 positive initial clone for 5 times, and adding 10ug/ml mycoplasma remover Plasmocin (Invivogen, ant-mpt) into the liquid for cloning; and finally, selecting 12 high-titer HPIV-3 clone strains derived from different HPIV-3 positive initial clones for exogenous factor detection, wherein the 12 clone strains are respectively named as LZ1501W 1-LZ 1501W 12.

1.3 detection of exogenous factors

Detecting rhinovirus, adenovirus, respiratory syncytial virus, influenza A H1N1, influenza A H2N3 and influenza B virus in an LZ1501 high-titer clone purified strain (LZ1501W 1-LZ 1501W12) by using a nested RT-PCR method, and detecting mycoplasma in the LZ1501 high-titer clone purified strain by using a nested RT-PCR method; the detection primer is designed by the second research room of the Limited responsibility company of Lanzhou biological product research institute and synthesized by Bao bioengineering (Dalian) Limited company; primer sequence information is shown in table 1 and verified by positive culture (fig. 2).

TABLE 1 detection primer sequences of exogenous factors

As a result, all LZ1501 high titer cloned purified strains were found to be negative for 7 exogenous factors (rhinovirus, adenovirus, respiratory syncytial virus, influenza A H1N1, influenza A H2N3 and influenza B virus, mycoplasma).

1.4 screening of HPIV-3 Cold-adapted temperature sensitive strains

Screening by adopting a mutagen and gradual cooling method, and specifically comprising the following steps: the 12 LZ1501 high-titer clone purified strains which are cloned and purified and have foreign factors removed are respectively inoculated into 12T 25 WI-38 cell culture flasks, 5-fluorouracil is added into each flask according to the concentration of 10ug/ml, and the ratio of the flasks is 1: 100 subculturing, wherein the culture temperature is 35 ℃, the temperature reduction range is 2 ℃, namely 35 ℃, 33 ℃, 31 ℃, 29 ℃, 27 ℃ and 25 ℃, and each temperature is subculturing for 10 generations; as a result, the average virus titer is reduced by 1lg value in 50 generations of cold adaptation subculture at 35-27 ℃; continuing to pass 35 generations at 25 ℃, and obviously reducing the virus titer of all clone culture wells to 4 lg values at most; further passaging was continued, as shown in FIG. 3, with the increase of the number of passages at 25 ℃ the titer of 2 clones decreased and then slowly increased to finally reach 10 after passage at 25 ℃ for 30 passages⁶Copy number/ml, one of the strains was named HPIV3CPW 3.

Example 2 preparation and purification of HPIV3CPW3 Virus culture fluid and identification of viral coat proteins

2.1 preparation of HPIV3CPW3 Virus culture solution

The HPIV3CPW3 strain was inoculated at 0.01MOI into WI-38 cells at 25 ℃ with 5% CO₂Culturing for 7 days in an incubator, collecting culture supernatant, detecting virus titer by qRT-PCR, and storing at 4 ℃.

2.2 ultracentrifugation purification of HPIV3CPW3 Virus

Centrifuging the HPIV3CPW3 virus culture solution prepared in the step 2.1 at 8000rpm and 4 ℃ for 30min, precipitating cell debris, and collecting supernatant; adding 30ml of supernatant into a 50ml special centrifuge tube, gently pushing 15ml of 50% sucrose from the bottom by using a 8cm long needle (the interface is kept clear in operation), placing the mixture into an ultracentrifuge (Hitachi cp70mx ultracentrifuge can be selected), ultracentrifuging for 4 hours at 4 ℃ and 35000rpm, and removing the supernatant; resuspending the precipitate with sterile TNE Buffer, determining the concentration of viral protein, and observing the virus morphology by electron microscope, wherein the virus particles with a size of about 150nm can be observed under electron microscope as shown in FIG. 4; the results show that the HPIV3CPW3 virus can be prepared in large scale, and the purified virus particles are clear and obvious, namely the HPIV3CPW3 virus is easy to purify and obtain.

2.3Western-Blot (Western immunoblotting) technique for the identification of the HN and F proteins of the virus coat of HPIV3CPW3

Carrying out SDS-PAGE electrophoresis on HPIV3CPW3 virus protein purified by ultracentrifugation, and carrying out Western-blotting analysis on the gel according to standard procedures (Sambrook J, Fritsch EF, Maniatis T1989) after the SDS-PAGE electrophoresis is finished;

wherein the specific antibodies are HN protein of rabbit anti-recombinant HPIV-3 and rabbit anti-recombinant F protein polyclonal antibody which are prepared by a second research laboratory of the Limited liability company of Lanzhou biological product research institute and are diluted according to a certain dilution for use; the secondary antibody is horseradish peroxidase (HRP) labeled goat anti-rabbit IgG antibody (purchased from Sigma company) and is used according to the labeled concentration; the color development was imaged using the Lumi-Light Western Blotting subsystem (chemiluminescent peroxidase substrate for Western Blotting, available from Roche1, model: 20152000010) using the GE Healthcare gel imaging system.

The results are shown in FIG. 5, in which the blot HN is the reaction band of HPIV3CPW3 virus HN protein and rabbit anti-recombinant HN polyclonal antibody, the blot F0 is the reaction band of HPIV3CPW3 virus F0 protein and rabbit anti-recombinant F protein polyclonal antibody, and the blot F1 is the reaction band of HPIV3CPW3 virus F1 protein and rabbit anti-recombinant F protein polyclonal antibody; as can be seen, the ultracentrifugally purified virus of step 2.2 has specific reaction bands with the HPIV-3 recombinant HN protein antibody and the HPIV-3 recombinant F protein antibody at the expected molecular weight, indicating that the HPIV3CPW3 strain has the HN protein and F protein of the HPIV-3 virus.

Example 3 determination of the Whole Gene sequence and analysis of genetic characteristics of HPIV3CPW3

The whole gene sequence of HPIV3CPW3 was determined by the second generation sequencing method, and the detailed procedure was completed by Shanghai Berger medical science and technology, Inc. The sequencing result shows that: the whole gene sequence of HPIV3CPW3 contains 15462 nucleotides, and the nucleotide sequence analysis shows that the homology of HPIV3CPW3 and HPIV-3 isolate MH678682.1 in GenBank is 99.87%, the nucleotide sequence of HPIV3CPW3 strain accords with the 6 base principle of paramyxoviridae genome, 6 proteins are encoded according to the sequence of 5 '-NP (111) -PP/C (1784) -3592) -M (3753) -4814) -F (5072) -6691) -HN (6806) -8524) -L (8571) -15347', and the gene structure is the same as that of the known sequence HPIV-3 isolate. Indicating that the HPIV3CPW3 virus strain is parainfluenza virus type iii.

Example 4 Cold-adapted and temperature-sensitive characterization of HPIV3CPW3 Virus in cell culture

Respectively inoculating HPIV3CPW3 and a parent wild strain LZ1501W3 thereof into a T25 cell culture bottle which is fully paved with WI-38 cells, respectively arranging three bottles, respectively culturing the three bottles of each virus at 25 ℃, 37 ℃ and 40 ℃ for 7 days, determining the titer of each bottle of virus culture solution according to 6.5qRT-PCR, and observing lesions;

as shown in fig. 6, HPIV3CPW3 significantly reduced lesions at 37 ℃ and 40 ℃ and significantly enhanced lesions at 25 ℃ compared to LZ1501W 3;

the virus titer test shows (fig. 7), the virus titer of the culture at 25 ℃ is improved by 3.59lg, which indicates that the HPIV3CPW3 has the characteristic of cold adaptation, and the virus titer of the culture at 40 ℃ is reduced by 4.86lg, the virus titer of the culture at 37 ℃ is reduced by 2.55lg, which indicates that the HPIV3CPW3 has the characteristic of temperature sensitivity.

Example 5 identification of temperature sensitive Properties of HPIV3CPW3 on a mouse model

Randomly dividing 2-week-old Balb/C mice with the weight of 13.5-14.5 g into two groups, wherein each group comprises 21 mice, and all the mice are anesthetized by intraperitoneal injection of 5% chloral hydrate and 100ul per mouse; after mice lost locomotion, one group of mice was inoculated intranasally with HPIV3CPW3 virus culture medium (titer 2.1X 10)⁶Copy/ml), 20 ul/piece; the other group of mice was inoculated nasally with the wild parent strain LZ1501 of HPIV3CPW3 strainW3 Virus culture fluid (titer 5.2X 10)⁸Copy/ml), 20 ul/piece; then, referring to the method of steps 6.4-6.5, the titer of HPIV-3 virus in mouse lung tissue and nasal wash was determined by qRT-PCR on day 3 after inoculation:

as shown in FIG. 8, the virus titers of LZ1501W3 and HPIV3CPW3 in lung tissue were 7.67lg and 5.58lg, respectively, which were significantly reduced by 2.09 lg; LZ1501W3 and HPIV3CPW3 increased titers in mouse nasal washes from 4.27lg to 4.78 lg by 0.51 lg. The results show that HPIV3CPW3 also has temperature-sensitive properties in the upper and lower respiratory tracts of Balb/C mice.

Example 6 preparation, immunization procedure and detection method of HPIV-3 attenuated live vaccine HPIV3CPW3

6.1 preparation of an attenuated live HPIV-3 vaccine

The virus culture solution of HPIV3CPW3 was inoculated into WI-38 cells at 25 deg.C in 5% CO2 incubator at 0.01MOI for 7 days, and the virus titer was 2.1X10 as determined by qRT-PCR⁶Copy/ml, 8000g for 40 min, and the centrifugation supernatant was collected as a vaccine to immunize mice.

6.2 immunization protocol and dose of HPIV3CPW3 immunized mice

Randomly dividing 2-week-old Balb/C mice with the weight of 13.5-14.5 g into 11 groups, wherein each group comprises 21 mice, and all the mice are anesthetized by intraperitoneal injection of 5% chloral hydrate and are 100 ul/mouse until the mice lose the power;

experimental group (HPIV3CPW3 group): 1-3 mice of the experimental group were vaccinated intranasally with the live attenuated HPIV-3 vaccine prepared in step 6.1 (HPIV3CPW3 virus broth, titer 2.1X 10)⁶Copy/ml), 20 ul/single, 1 dose of total inoculation; 4-6 experiment groups, the nasal cavity is inoculated with 20ul of HPIV-3 attenuated live vaccine, 2 doses are inoculated, and the interval of each dose is 28 days; 7-9 experiment groups, the nasal cavity was inoculated with HPIV-3 attenuated live vaccine, 20 ul/mouse, 3 doses were administered, with 28 days intervals.

Positive control group (LZ1501W3 group): compared with the experimental group, the difference is that the anesthetized mice of the 10 th group are inoculated with the virus culture solution (titer 5.2X 10) of the wild parental strain LZ1501W3 of HPIV3CPW3 virus through nasal cavity⁸Copy/ml), breeding separately with experimental group, inoculating only one dose;

negative control group: compared with the experimental group, the difference is that 20ul of high-sugar DMEM liquid culture medium is inoculated to the anesthetized mice of the 11 th group, and the anesthetized mice are separately raised with the experimental group without any treatment;

6.3 harvesting of upper and lower respiratory tissues from HPIV3CPW3 infected mice

Experimental groups: respectively taking a group of mice on the 3 rd day and the 40 th day after each dose of inoculation (for the 1 st to 3 rd experimental groups, a group of HPIV3CPW3 infected mice on the 3 rd day and the 40 th day after 1 dose of inoculation in the three groups, for the 4 th to 6 th experimental groups, a group of mice on the 3 rd day and the 40 th day after 2 doses of inoculation in the three groups, and for the 7 th to 9 th experimental groups, a group of CPW3 infected mice on the 3 rd day and the 40 th day after 3 doses of inoculation in the three groups); 500ul of whole blood is taken from the eyeballs of mice infected by HPIV3CPW3 in each group, the whole blood is kept still at 37 ℃ for 2 hours, 5000g of whole blood is centrifuged for 10 minutes after 4 ℃ is over night, and serum is separated and used for measuring the titer of the neutralizing antibody; then, the neck is pulled to kill the infected mouse, the nasal cavity of the mouse is washed by 100ul of high-sugar DMEM liquid culture medium, 50ul of washing liquid is taken to extract RNA, and the RNA is used for qRT-PCR detection of the titer of the HPIV-3 virus in the washing liquid of the nasal cavity of the mouse; soaking the mice in 75% ethanol for 2 minutes for sterilizing, aseptically debriding and infecting the chest cavities of the mice, taking out the lungs, weighing 10mg, grinding by using a 1ml glass homogenizer, and extracting RNA for qRT-PCR detection of the HPIV-3 virus titer in the lung tissues of the mice.

6.4 extraction of RNA from upper and lower respiratory tissues of HPIV3CPW 3-infected mice

The RNA extraction of mouse nasal wash and lung tissue RNA extraction were performed with Rneasy Mini Kit (50) (QIAGEN, 74104), as follows: extracting RNA from mouse nasal wash: taking 50ul of mouse nasal wash, adding 110ul of sterilized PBS, adding 150ul of buffer RLT and 3ul of beta-ME into the mouse nasal wash, and thoroughly mixing the mouse nasal wash and the PBS; adding 300ul 70% ethanol (prepared with DEPC water), and mixing; adding the mixture into an RNA purification column, centrifuging at 12000rpm for 1min, and discarding the liquid in the tube; adding 700uL buffer RW1, centrifuging at 12000rpm for 1min, and discarding the liquid in the tube; adding 500uL buffer RPE, centrifuging at 12000rpm for 1min, and discarding the liquid in the tube; adding 500uL buffer RPE, washing again, and discarding the liquid in the tube; centrifuging at 12000rpm for 2min, and emptying; replacing the centrifuge tube, adding 20uL DEPC water into each column, and centrifuging at 12000rpm for 1 min; the purified RNA was placed in an ice bath or at-80 ℃ until use.

Extraction of mouse lung tissue RNA: adding 10mg mouse lung tissue into 800ul buffer RLT (10ul beta-ME/1 ml RLT solution), crushing the tissue with a grinding rod, centrifuging at 12000rpm for 10min, taking 300ul supernatant into a new EP tube, adding 300ul 70% ethanol (prepared by DEPC water), and mixing; adding the mixture into an RNA purification column, centrifuging at 12000rpm for 1min, and discarding the liquid in the tube; adding 700uL buffer RW1, centrifuging at 12000rpm for 1min, and discarding the liquid in the tube; adding 500uL buffer RPE, centrifuging at 12000rpm for 1min, and discarding the liquid in the tube; adding 500uL buffer RPE, washing again, and discarding the liquid in the tube; centrifuging at 12000rpm for 2min, and emptying; replacing the centrifuge tube, adding 20uL DEPC water into each column, and centrifuging at 12000rpm for 1 min; the purified RNA was placed in an ice bath or at-80 ℃ until use.

6.5 detection of viral titer of HPIV-3 in the Upper and lower respiratory tracts of mice

The method for detecting the virus titer of the HPIV3CPW3 infected mouse upper and lower respiratory tract HPIV-3 by adopting a qRT-PCR method comprises the following steps:

6.5.1 primer and Probe design for the qRT-PCR method for the detection of the Virus titer of HPIV-3

The primers and the probes are positioned in the HN segment of HPIV-3, are designed and verified by the laboratory, and the sequences are shown in the table 2;

TABLE 2 primer and Probe sequences for the detection of HPIV-3 Titers Using the Q-RT-PCR method

6.5.2 preparation of HNRNA standard for qRT-PCR detection

The in vitro transcription method is adopted, and the specific steps are as follows:

6.5.2.1 cloning of HPIV-3HN Gene

Cloning of HPIV-3HN Gene by RT-PCR: HPIV-3 viral RNA was extracted according to the method of 6.4, and HPIV 3HN gene was amplified using SuperScript III One Step RT-PCR system with platinum Taq High Fidelity (Invitrogen, 12574-035) and primers HNS (ATGGAATACTGGAAGCACCC)/HNA (AACTGCAGCTTTTTGGAAT);

reaction system: 2x reaction buffer 25ul, RNA 5ul, HNS (10pmol/ul)1ul, HNA (10pmol/ul)1ul, SuperScript III RT/Taq En 1ul,DEPC H₂O 17ul；

Reaction procedure: 30 minutes at 48 ℃; 2 minutes at 94 ℃; repeating the steps of 94 ℃ for 40 seconds, 55 ℃ for 1 minute and 68 ℃ for 2 minutes for 30 times; 8 minutes at 68 ℃;

the RT-PCR product was subjected to Gel electrophoresis, the band of interest was excised, and HN DNA was purified using QIAquick Gel Extraction Kit according to the method indicated in the Kit. The sequencing result shows that the sequence homology with the HPIV-3HN gene on GenBank is 99.81 percent, which indicates that the HPIV-3HN gene is successfully cloned.

6.5.2.2 preparation of HPIV-3HNRNA standard

The T7 RNA polymerase kit (purchased from Takara, D2540) was used to prepare standards by the following procedures: using the HPIV-3HN gene obtained from 6.5.2.1 as a template, and using HNT7S and HNT7A primers (see Table 3) to amplify an HPIV-3HN gene fragment with a T7 RNA polymerase initiation site at the 5 'end and Poly (A)15 at the 3' end; after the fragment is cut and purified, the fragment is used as an in vitro transcription template, and the RNA of HPIV-3HN is obtained by in vitro transcription by using a T7 RNA polymerase kit; after in vitro transcription HPIV-3HN RNA is purified, the purity and the concentration are determined, and the purified product is used as a standard product of a qRT-PCR detection method after 10x serial dilution to determine a standard curve.

TABLE 3HPIV-3 HN RNA in vitro transcription primer sequences

6.5.3HPIV-3 Virus titer detection system and program

Adopt One Step PrimeScript^TMHPIV-3 virus titer detection was performed by RT-PCR Kit (Perfect Real Time) (Takara, Code number DRR064A) by the following specific procedures:

using 3.7x10⁰-3.7x10⁷Copying/ml of in vitro transcription HPIV-3HN RNA, performing Taqman probe Real-Time RT-PCR, and establishing a standard curve;

the reaction system is as follows: 2xone step RT-PCR buffer III 10ul, Takara Ex TaqHS (5U/ul)0.4ul, PrimeScript RT Enzyme Mix II 0.4ul, HNReaLS (10pmol/ul)0.6ul, HNReaLA (10pmol/ul)0.6ul, Probe (10pmol/ul)0.2ul, ROX Reference Dye (50x)0.4ul, Tatol RNA 2ul, RNase free 205H 4ul, total volume 20 ul;

the amplification procedure was: 1. reverse transcription at 42 deg.c for 5 min and 95 deg.c for 10 sec; 2. PCR amplification, 95 ℃ for 5 seconds, 57 ℃ for 34 seconds, 40 cycles; the above amplification reaction was carried out on a 7500real-time PCR system (Applied Biosystem).

6.6 detection of neutralizing antibodies to HPIV-3 in serum of mice immunized with HPIV3CPW3

The plaque reduction test is adopted to detect the titer of the HPIV-3 neutralizing antibody of the serum of the immunized mouse, and the specific operation is as follows:

6.6.1 plaque assay determination of working dilution with the virus HPIV3LZ1728C19

Diluting HPIV-3 wild strain HPIV3LZ1728C19 in 5 × series, inoculating 24-well plate MA104 cell, standing at 37 deg.C and 5% CO₂Culturing for 2 hr in incubator, sucking out supernatant, adding 1% low melting point agarose 3ml, coagulating at room temperature, and pouring at 37 deg.C with 5% CO₂Culturing in incubator for 72 hr, adding 1ml of 1% low melting point agarose containing neutral red, solidifying at room temperature, and cooling at 37 deg.C with 5% CO₂The incubator stays overnight, spots are observed, and the virus dilution with 80% of cells having spots is taken as the working dilution of the virus;

6.6.2 dilution of the serum to be tested and neutralization of the Virus

Diluting the serum to be detected (HPIV3CPW3 immune mouse serum) 10 times, sterilizing and filtering with 0.22 μ M needle filter, diluting 2 × series, mixing with HPIV3LZ1728C19 virus diluted with equal volume of working dilution, and combining at 37 deg.C for 2 hr; inoculating virus neutralized by serum into MA104 cell of 24-well plate, and placing at 37 deg.C and 5% CO₂Culturing for 2 hr in incubator, sucking out supernatant, adding 1ml of 1% low melting point agarose, coagulating at room temperature, and inverting at 37 deg.C and 5% CO₂Culturing in incubator for 72 hr, adding 1ml of 1% low melting point agarose containing neutral red, solidifying at room temperature, and cooling at 37 deg.C with 5% CO₂Overnight in the incubator, spots were observed (experimental group);

6.6.3 negative control

The negative control serum was diluted 20 Xand mixed with an equal volume of the working dilution of HPIV3LZ1728C19 virus, and the rest steps were as in 6.6.2;

6.6.4 judging the result: the serum dilution which reduced the plaque number by 50% compared to the plaque number of the negative control group was determined as the neutralizing antibody titer (fig. 9).

Example 7 evaluation of the safety, immunogenicity and protective efficacy of HPIV3CPW3 immunized mice

7.1 proliferation of HPIV3CPW3 infected mice in the upper and lower respiratory tract

Experimental groups: taking the experimental group mice in step 6.2 of example 6 as the study objects, extracting mouse nasal wash and lung tissue RNA by the method of steps 6.3 and 6.4 on the 3 rd day after each dose of inoculation, and detecting the HPIV3CPW3 virus load of the immunized mice by the qRT-PCR method by the method of step 6.5;

negative control group: compared with the experimental group, the difference is that each anesthetized mouse is inoculated with 20ul of high-sugar DMEM liquid culture medium, and the anesthetized mouse is bred separately from the experimental group without any treatment;

as shown in fig. 10, HPIV3CPW3 was significantly proliferated in the upper and lower respiratory tracts of mice after each dose of inoculation, compared to the negative control group; 3 days after one dose of immunization, the virus load of HPIV3CPW3 in the lung of the mouse is improved by 2.7lg, and the virus load in the nasal cavity is 3.1 lg; the above results indicate that the HPIV3CPW3 virus strain can infect mice and proliferate in the upper and lower respiratory tracts of the mice

7.2 evaluation of safety of mice immunized with HPIV3CPW3

Taking the mice in the experimental group in the step 6.2 of the example 6 as research objects, and continuously measuring the body weight and the body temperature of the mice in the 1 st group, the positive control group and the negative control group in the experimental group for 30 days;

the results are shown in fig. 11, the weight curve of the immunized mouse within 30 days is basically coincident with that of the negative control group, and no difference exists; the body temperature of the mice fluctuated within the normal range within 30 days after the immunization, which indicates that the safety of the HPIV3CPW3 to the mice is good.

7.3 evaluation of neutralizing antibody response in sera of mice immunized with HPIV3CPW3

Experimental groups: using the experimental group of mice in step 6.2 of example 6 as subjects, a group of HPIV3CPW3 infected mice were taken on day 40 after each dose of inoculation, and the ability of HPIV3CPW3 to induce the production of neutralizing antibodies in the mice was tested using the plaque reduction neutralization assay of step 6.6 of example 6;

negative control group (group 11 in step 6.2): compared with the experimental group, the difference is that each anesthetized mouse is inoculated with 20ul of high-sugar DMEM liquid culture medium, and the anesthetized mouse is bred separately from the experimental group without any treatment;

as shown in fig. 12, the geometric mean titer of the antibody in the mice after 2-dose immunization was increased 103-fold and the geometric mean titer of the antibody in the mice after 3-dose immunization was increased 1386-fold, compared with the negative control group; it is demonstrated that HPIV3CPW3 immunized mice can induce a significant serum HPIV-3 specific neutralizing antibody response with a dose-by-dose effect.

7.4 evaluation of the protective efficacy of HPIV3CPW3 immunization on HPIV-3 infection in the lungs of mice

Experimental groups: taking the experimental group of mice in step 6.2 of example 6 as the study object, a group of HPIV3CPW3 infected mice were taken at the 40 th day after each dose of inoculation and used for the HPIV-3 infection challenge experiment, which specifically comprises the following steps: using the wild strain HPIV-3 HPIV3LZ1728C19(8.5X 10)⁸Copy/ml) infected HPIV3CPW3 immune mice by nasal cavity challenge, 20 ul/mouse, collecting samples and extracting RNA according to the steps 6.3-6.4 on the third day after the challenge, and detecting the virus titer of HPIV-3 in the samples by using a qRT-PCR method according to the step 6.5;

negative control group (group 11 in step 6.2): compared with the experimental group, the difference is that 20ul of DMEM liquid culture medium is inoculated to each anesthetized mouse in the 11 th group, and the anesthetized mice are separately raised with the experimental group without any treatment;

the results are shown in fig. 13, and the challenge experiment of HPIV3LZ1728C19 shows that the virus load of mouse lung HPIV3LZ1729C19 is significantly reduced by 0.81lg (p <0.05) after 2 doses of vaccination and the virus load of mouse lung is reduced by 1.85lg (p <0.005) after 3 doses of vaccination compared with the negative control group, which indicates that HPIV3CPW3 immunization has significant protective efficacy on infection of mouse lung HPIV-3 wild strain HPIV3LZ1728C 19.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.

Sequence listing

<110> limited liability company of research institute for biological products of Lanzhou

<120> human triple parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA