阅读说明：本技术 柯萨奇病毒a组16型单克隆抗体16e1及其制备方法 (Coxsackie virus A group 16 type monoclonal antibody 16E1 and preparation method thereof ) 是由 程通 叶祥忠 毛群颖 徐龙发 梁争论 高帆 韩金乐 夏宁邵 于 2019-10-30 设计创作，主要内容包括：本发明提供一种柯萨奇病毒A组16型单克隆抗体16E1,所述单克隆抗体是由杂交瘤细胞株16E1产生,该杂交瘤细胞株于2019年8月1日保藏于中国典型培养物保藏中心,保藏编号为CCTCC No.C2019167。本发明的16E1单克隆抗体具备良好的特异性,不与肠道病毒的其他型别发生交叉反应,对不同来源的CA16病毒具有良好的广谱性,具有优秀的应用意义。(The invention provides a Coxsackie virus A group 16 type monoclonal antibody 16E1, which is generated by a hybridoma cell strain 16E1, wherein the hybridoma cell strain is preserved in a China center for type culture Collection in 2019, 8 and 1 months, and the preservation number is CCTCC No. C2019167. The 16E1 monoclonal antibody has good specificity, does not have cross reaction with other types of enteroviruses, has good broad spectrum for CA16 viruses of different sources, and has excellent application significance.)

1. The Coxsackie virus A group 16 type monoclonal antibody 16E1 is produced by a hybridoma cell strain 16E1, and the hybridoma cell strain is preserved in the China center for type culture collection at 8 months and 1 days in 2019, wherein the preservation number is CCTCC No. C2019167, and the preservation address is Wuhan university, Wuhan, China.

2. The method for constructing hybridoma cell line 16E1 according to claim 1, comprising the following steps:

(1) immunization of mice

Immunizing a BALB/c female mouse with the age of 6-8 weeks by using a CA16 virus immunogen, strengthening the immunization once every 2 weeks, detecting the serum titer of the mouse before each immunization, stopping the immunization when the serum titer of the mouse reaches a platform period, and preparing for fusion;

(2) preparation and screening of fused hybridomas

(2.1) preparation of mouse macrophage

Killing BALB/c mouse, dissolving abdominal cavity cell liquid in HAT culture solution or HT culture solution until the final concentration of cells in the culture solution is 2 × 10⁵Per mL; adding the cells into a 96-hole cell culture plate, and culturing in an incubator to obtain mouse macrophages;

(2.2) preparation of mouse thymocytes

Killing BALB/c mice, taking out thymus, grinding the thymus, and sieving through a 200-mesh cell sieve to obtain thymus feeder cell liquid;

(2.3) preparation of mouse myeloma cells

Taking mouse myeloma cell strain Sp2/0-Ag14(Sp2/0), culturing with 20% FBS RPMI-1640 culture solution, selecting cells in logarithmic growth phase, transferring myeloma cells from a culture bottle into a centrifuge tube before fusion, washing with RPMI-1640 culture solution for 3 times, resuspending the cells with the RPMI-1640 culture solution, and counting;

(2.4) preparation of immune spleen cells

Taking a BALB/C mouse to be fused, and collecting blood after the mouse is sacrificed to prepare antiserum serving as a positive control of antibody detection; taking mouse spleen, sieving with 200 mesh cell sieve, squeezing and grinding with grinding rod, dripping RPMI-1640 culture solution, standing for 3-5min, taking 2/3 part of suspension, and transferring into 50mL plastic centrifuge tube; repeating the steps for 2-3 times, washing the cells for 3 times by using RPMI-1640 culture solution, then re-suspending the cells by using the RPMI-1640 culture solution and counting to obtain immune spleen cells;

(2.5) preparation of hybridomas Using PEG fusion promoters

1mL of PEG-1500, 10mL of RPMI-1640 serum-free medium, and 200mL of complete medium were equilibrated to 37 ℃; mixing the myeloma cells prepared in the step (2.3) and the spleen cells prepared in the step (2.4) in a 50mL centrifugal tube, wherein the number ratio of the spleen cells to the myeloma cells is 10:1, centrifuging at 1500rpm for 8min, and lightly tapping the bottom of the tube to loosen the cells into paste; sucking 0.8mL of PEG, adding into a centrifugal tube, adding 10mL of RPM-1640 complete culture solution at 37 ℃, stirring gently, finally supplementing the RPMI-1640 culture solution to 40mL, and centrifuging at 1000RPM for 5 min; discarding the supernatant, taking the HT culture solution to blow the cells away, and then adding the HT culture solution; adding the obtained cells into a 96-well cell culture plate, wherein each well is 100 mu L, putting the cell culture plate into a CO2 incubator for 12h, and then dropwise adding 100 mu L HAT complete culture medium into each well; after culturing for 5 days in a CO2 incubator, using an HT complete culture medium to exchange liquid for cell supernatant in the holes, wherein the volume ratio of the exchange liquid is 50-100%; after 9-14 days, sucking the supernatant for detection;

(2.6) screening of hybridomas

Screening by using an indirect ELISA method, coating 100 ng/hole of purified CA16 virus antigen, adding 50 mu L of cell supernatant for detection, and selecting a positive cloning hole;

(2.7) cloning of hybridoma cells

And selecting hybridoma cells by using a clone culture method, and repeatedly cloning for 2-3 times until the hybridoma cells are 100% positive to obtain the hybridoma cells 16E 1.

3. The monoclonal antibody 16E1 of claim 1, for use in detection of coxsackievirus A16 type virus antigen.

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of virology and antibody medicines, in particular to a coxsackie virus A group 16 type monoclonal antibody 16E1, a hybridoma cell strain for generating the monoclonal antibody, and a construction method and application of the cell strain.

[ background of the invention ]

The hand-foot-and-mouth disease is one of class-C infectious diseases in China, the first occurrence of the disease is fever, rash, herpes and small ulcer of hands, feet and oral cavities, and part of patients can develop aseptic encephalitis, meningitis and other serious disease manifestations, so that a large disease burden is brought to China. Currently, most areas around the world have reports related to hand-foot-and-mouth diseases, and the asia-pacific area is regarded as a high-incidence area of hand-foot-and-mouth diseases and receives enough attention in recent years. Enterovirus 71 (EV71) and coxsackievirus A group 16 (CA16) are main pathogens causing hand-foot-and-mouth disease, and although the hand-foot-and-mouth disease caused by coxsackievirus A group 6 (CA6) and coxsackievirus A group 10 (CA10) is increasing in recent years, EV71 and CA16 infection has obvious association with severe hand-foot-and-mouth disease.

CA16 is a member of Enterovirus of picornaviridae, has a diameter of 27-30 nm, is non-enveloped, and has an icosahedral structure. The single-strand positive-strand RNA virus has a 7400bp genome with an open reading frame, encodes a polyprotein precursor which is cleaved into a structural protein P1 and non-structural proteins P2 and P3 by self-produced hydrolases. The structural protein P1 mainly encodes capsid protein composed of VP1, VP0 and VP3, VP0 is hydrolyzed into VP2 and VP4 in the virus maturation process, wherein VP4 is located in the capsid protein, and VP1, VP2 and VP3 are distributed on the surface of capsid virus to form pentamer. The open reading frame coding region is flanked on both sides by 5 'and 3' non-coding regions, respectively, the 5 'non-coding region consisting of 740 nucleotides and being associated with replication and translation functions of the viral genome, and the 3' non-coding region comprising a PolyA tail, which is essential for successful infection of the virus.

The detection of the content of the virus antigen is an important means for quality control in the vaccine production process, but a unified CA16 antigen detection kit is still lacked at present. The CA16 monoclonal antibody is an important component for ensuring the specificity and accuracy of the kit.

[ summary of the invention ]

The invention aims to overcome the defect of the blank CA16 antigen detection system, and provides a CA16 monoclonal antibody, and an in-vitro detection method and a detection kit for a CA16 antigen are established by applying the monoclonal antibody, wherein the kit has broad spectrum, can react with other types of antigens of CA16, has good specificity and does not have cross reaction with viruses such as EV71, CA6, CA10 and the like and other types of viruses.

In order to achieve the purpose, the invention provides a monoclonal antibody 16E1 of a coxsackievirus A16 type virus, which is produced by a hybridoma cell strain 16E1, wherein the hybridoma cell strain is preserved in a China center for type culture Collection in 8.1.2019, the preservation number is CCTCC number C2019167, and the preservation address is university of Wuhan, China.

The invention also provides a construction method of the hybridoma cell 16E1, which comprises the following steps:

(1) immunization of mice

Immunizing a BALB/c female mouse with the age of 6-8 weeks by using a CA16 virus immunogen, strengthening the immunization once every 2 weeks, detecting the serum titer of the mouse before each immunization, stopping the immunization when the serum titer of the mouse reaches a platform period, and preparing for fusion;

(2) preparation and screening of fused hybridomas

(2.1) preparation of mouse macrophage

Killing BALB/c mouse, dissolving abdominal cavity cell liquid in HAT culture solution or HT culture solution until the final concentration of cells in the culture solution is 2 × 10⁵Per mL; adding the cells into a 96-hole cell culture plate, and culturing in an incubator to obtain mouse macrophages;

(2.2) preparation of mouse thymocytes

Killing BALB/c mice, taking out thymus, grinding the thymus, and sieving through a 200-mesh cell sieve to obtain thymus feeder cell liquid;

(2.3) preparation of mouse myeloma cells

Taking mouse myeloma cell strain Sp2/0-Ag14(Sp2/0), culturing with 20% FBS RPMI-1640 culture solution, selecting cells in logarithmic growth phase, transferring myeloma cells from a culture bottle into a centrifuge tube before fusion, washing with RPMI-1640 culture solution for 3 times, resuspending the cells with the RPMI-1640 culture solution, and counting;

(2.4) preparation of immune spleen cells

Taking a BALB/C mouse to be fused, and collecting blood after the mouse is sacrificed to prepare antiserum serving as a positive control of antibody detection; taking mouse spleen, sieving with 200 mesh cell sieve, squeezing and grinding with grinding rod, dripping RPMI-1640 culture solution, standing for 3-5min, taking 2/3 part of suspension, and transferring into 50mL plastic centrifuge tube; repeating the steps for 2-3 times, washing the cells for 3 times by using RPMI-1640 culture solution, then re-suspending the cells by using the RPMI-1640 culture solution and counting to obtain immune spleen cells;

(2.5) preparation of hybridomas Using PEG fusion promoters

1mL of PEG-1500, 10mL of RPMI-1640 serum-free medium, and 200mL of complete medium were equilibrated to 37 ℃; mixing the myeloma cells prepared in the step (2.3) and the spleen cells prepared in the step (2.4) in a 50mL centrifugal tube, wherein the number ratio of the spleen cells to the myeloma cells is 10:1, centrifuging at 1500rpm for 8min, and lightly tapping the bottom of the tube to loosen the cells into paste; sucking 0.8mL of PEG, adding into a centrifugal tube, adding 10mL of RPM-1640 complete culture solution at 37 ℃, stirring gently, finally supplementing the RPMI-1640 culture solution to 40mL, and centrifuging at 1000RPM for 5 min; discarding the supernatant, taking the HT culture solution to blow the cells away, and then adding the HT culture solution; the resulting cells were added to a 96-well cell culture plate at 100. mu.L per well, and CO was added₂After 12h of incubation in the incubator, 100 μ L of HAT complete medium was added dropwise to each well; after culturing for 5 days in a CO2 incubator, using an HT complete culture medium to exchange liquid for cell supernatant in the holes, wherein the volume ratio of the exchange liquid is 50-100%; after 9-14 days, sucking the supernatant for detection;

(2.6) screening of hybridomas

Screening by using an indirect ELISA method, coating 100 ng/hole of purified CA16 virus antigen, adding 50 mu L of cell supernatant for detection, and selecting a positive cloning hole;

(2.7) cloning of hybridoma cells

And selecting hybridoma cells by using a clone culture method, and repeatedly cloning for 2-3 times until the hybridoma cells are 100% positive to obtain the hybridoma cells 16E 1.

The invention also provides application of the monoclonal antibody 16E1 in detection of coxsackie virus A16 type virus antigen.

Experiments prove that the 16E1 monoclonal antibody has no cross reaction with other types of enteroviruses and has good specificity. In addition, the monoclonal antibody can react with antigens of a plurality of subtypes of CA16, and has broad spectrum.

Because the enteroviruses have very obvious intra-type and inter-type cross reactivity, for example, the similarity of genomes of EV71 and CA16 reaches 80%, while the monoclonal antibody 16E1 does not have cross reaction with enteroviruses of other types such as EV71, CA6, CA10 and the like, so that the restriction of the cross reactivity on the establishment of a kit can be well avoided, and the application significance is excellent.

[ description of the drawings ]

FIG. 1 is an SDS-PAGE electrophoresis of CA16 rabbit polyclonal antiserum from example 1 after purification,

wherein lane 1: pre-staining protein marker; lane 2: and (5) purifying the polyclonal antibody.

Figure 2 is the sensitivity and linear range results of example 6.

FIG. 3 shows the accuracy results of example 6.

FIG. 4 shows the results of the applicability study of example 7.

[ detailed description ] embodiments

The following examples serve to illustrate the technical solution of the present invention without limiting it.

The following examples relate to the following materials or devices, which are also directly available to the skilled person in the light of the prior art:

the following examples relate to the following materials or devices:

the sources of the CA16 virus were:

(1) the strain CA16V-24 is provided by Beijing Kexing biological products, Inc., and can be obtained by the same method by those skilled in the art, and the strain specimen is from Hangzhou city, Zhejiang province.

(2) CA16/00190/B1B (GenBank No. MF177223) virus was isolated from Xiamen university in 2011 and specimens were obtained from Taiwan clinical samples positive for HFMD.

The RPMI-1640 culture solution comprises the following components: dry powder of medium (GIBCO-31800-.

FBS：GIBCO-10099-141

The 20% FBS RPMI-1640 culture solution is prepared by adding 20% FBS by total volume on the basis of the RPMI-1640 culture solution.

The HT culture solution is prepared by adding hypoxanthine (SIGMA-ALBRICH-H9377) and thymidine (SIGMA-ALBRICH-T9250) to RPMI-1640 culture solution.

HAT culture medium is HT culture medium supplemented with aminopterin (SIGMA-ALBRICH-A5159).

The RPMI-1640 complete culture solution is prepared by adding 10% FBS (FBS) into the RPMI-1640 culture solution according to the total volume.

HT complete medium is obtained by adding 10% FBS to HT medium in total volume.

HAT complete medium is prepared by adding 10% FBS to HAT medium.

In the present invention, "%" used for explaining the concentration is mass percent, ": all the terms are by weight.