CN108531442B - Insect cell line cultured in serum-free suspension manner and application thereof - Google Patents
Insect cell line cultured in serum-free suspension manner and application thereof Download PDFInfo
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Abstract
The invention discloses an insect cell line, which is derived from an embryonic cell line QAU-Tn-E-7 of Trichoplusia ni, wherein the preservation number of the cell line is CCTCC NO: C201865. The cell line of the invention is suitable for serum-free suspension culture, does not contain TNCL virus, has high growth speed, high cell density and high virus yield and protein expression level under the serum-free suspension culture condition, and is an ideal cell line for producing insect virus insecticide and exogenous recombinant protein in a large scale.
Description
Technical Field
The invention belongs to the field of agriculture and biotechnology, and particularly relates to a serum-free suspension culture insect cell line and application thereof, namely a Trichoplusia ni embryo cell line QAU-Tn-E-7, which is suitable for large-scale production of insect virus insecticides and exogenous recombinant proteins.
Background
Insect cell lines play very important roles in the production of baculovirus, the expression of recombinant protein and the like, and insect cell culture media generally contain a certain proportion of animal serum to support the growth and proliferation of cells, but the application of the insect cell culture media is limited due to the defects that the serum is high in cost and complex in components, and the separation, purification and detection of a culture product at a later stage are difficult and the like. Research and development in the fields of cell engineering, genetic engineering, protein engineering, medical biology and the like urgently need the support of a serum-free cell culture technology, so that the serum-free culture of insect cells is increasingly emphasized by people and becomes a new hotspot in the field of cell culture engineering. Meanwhile, most insect cell lines grow in an adherent manner, and the density of cells and the production yield are difficult to reach the highest due to the limitation of an adherent area, so that the large-scale production is limited.
Currently, the most internationally applied insect cell lines in the aspects of baculovirus production, exogenous recombinant protein expression and the like are Sf-21 of Spodoptera frugiperda or a clone Sf-9 thereof and BTI-Tn5B1-4(High Five) of Trichoplusia ni, and the Sf-9 and BTI-Tn5B1-4 cell lines have advantages and disadvantages in practical application, and the two cell lines have High growth speed under the adherent condition, but the cells are easy to agglomerate during suspension culture to influence the growth of the cells. The titer of the insect baculovirus forming the geminivirus (BV) in Sf-9 cells is higher, but the yield of virus polyhedra and the expression amount of recombinant protein are lower than those of BTI-Tn5B1-4 cells. In addition, a TNCL (Tn5cell line) virus is detected in BTI-Tn5B1-4 cells, which belongs to the family of Nodaviridae (Nodaviridae), the genus alpha-nodavirus (Alphanodavirus), and which is capable of infecting suckling mice and baby hamsters and causing paralysis and death thereof, so that BTI-Tn5B1-4 cells need to be tested for the presence of this virus at the time of use in order to reduce the potential risk of BTI-Tn5B1-4 cells in the production of medical drugs and vaccines (Li T C, et al, tension infection of a new Alphanodavirus in an infection cell line, Journal of virology,2007,81(20): 10890-. Therefore, aiming at the defects, the screening and obtaining of a new cell line which is free of TNCL virus, suitable for serum-free suspension culture and has large-scale industrial culture potential has wide application prospect.
Disclosure of Invention
The invention provides an insect cell line which is suitable for serum-free suspension culture and does not contain TNCL virus, the cell line has high growth speed, high cell density and high virus yield and protein expression level under the serum-free suspension culture condition, and is suitable for the large-scale production of insect virus insecticides and exogenous recombinant proteins.
The insect cell line provided by the invention is a Trichoplusia ni cell line QAU-Tn-E-7(Trichoplusia ni cell line QAU-Tn-E-7), which is preserved in the China center for type culture Collection in Wuhan university and Wuhan university in 2018, 4 and 17 days, and the preservation number is CCTCC NO: C201865.
The cell line is directly used for Sf-900TMIII SFM serum-free medium is used for carrying out primary culture and subculture on the embryo cells of the Trichoplusia ni, the established cell line with stable growth is obtained by continuously subculturing for 50 generations and screening, and the growth state of the cells is good.
The cell line provided by the invention does not contain TNCL virus. TNCL virus fragments were detected from BTI5B1-4 cells by reverse transcription PCR (RT-PCR) detection, while fragments of the corresponding size were not amplified in QAU-Tn-E-7 cells, demonstrating that the QAU-Tn-E-7 cells do not contain TNCL virus.
The cell line provided by the invention can be used for preparing virus insecticides, is highly sensitive to viruses and has high yield of virus polyhedrosis, wherein the viruses are Autographa californica nuclear polyhedrosis viruses (AcMNPV). The infection rate of AcMNPV of the cells cultured in a suspension culture flask (spinner flash) can reach 100 percent, the average yield of virus polyhedra per cell is 106.2, and the yield of virus polyhedra can reach 3.12 multiplied by 108PIB/mL。
The cell line provided by the invention canIs used for expressing and producing exogenous recombinant protein, such as beta-galactosidase (beta-gal) and alkaline phosphatase (SEAP). The recombinant protein expression level of the cells cultured in a suspension culture flask (spinner flash) is obviously improved, and the beta-galactosidase expression level reaches 3.61 multiplied by 104IU/mL, the activity of alkaline phosphatase reaches 3.97 IU/mL.
The invention provides an insect cell line (Trichoplusia ni cell line QAU-Tn-E-7) suitable for serum-free suspension culture, which has the advantages of high growth speed, high cell density, high virus yield and high protein expression level under the serum-free suspension culture condition, and has wide application prospect in the aspects of large-scale production of insect virus insecticides and exogenous recombinant proteins and the like.
Drawings
FIG. 1 is a morphological diagram of cell line QAU-Tn-E-7 in serum-free medium;
FIG. 2 is a diagram of RT-PCR for detecting cell line TNCL virus;
FIG. 3 is a graph of growth curves and viability for cell lines; wherein A is BTI-Tn5B1-4(High Five) cell at 25cm2Culturing in culture flask with TNM-FH medium containing 10% FBS, wherein B is QAU-Tn-E-7 cells at 25cm2Sf-900 used in culture bottleTMIII SFM Medium, QAU-Tn-E-7 cells in 125mL suspension culture flasks with Sf-900TMIII SFM culture medium.
FIG. 4 is a diagram of a cell line infected with Autographa californica nuclear polyhedrosis virus (AcMNPV);
FIG. 5 is a graph showing the expression level of the recombinant protein β -galactosidase (. beta. -gal) of the cell line; wherein A is BTI-Tn5B1-4(High Five) cell at 25cm2Culturing in culture flask with TNM-FH medium containing 10% FBS, wherein B is QAU-Tn-E-7 cells at 25cm2Sf-900 used in culture bottleTMIII SFM Medium, QAU-Tn-E-7 cells in 125mL suspension culture flasks with Sf-900TMIII SFM culture medium.
FIG. 6 is a graph showing the expression levels of the cell line recombinant protein alkaline phosphatase (SEAP); a is BTI-Tn5B1-4(High Five) cell at 25cm2The culture flask is cultured by TNM-FH medium containing 10% FBS, and B is QAU-Tn-E-7 cells25cm2Sf-900 used in culture bottleTMIII SFM Medium, QAU-Tn-E-7 cells in 125mL suspension culture flasks with Sf-900TMIII SFM culture medium.
Detailed Description
The invention cultures the Trichoplusia ni embryonic cells to establish a Trichoplusia ni cell line, obtains a suspension cell line QAU-Tn-E-7 which has stable growth and is sensitive to viruses by screening, and has the following preservation number: CCTCC NO of C201865; the preservation time is as follows: year 2018, month 4, day 17; the storage unit is: china center for type culture Collection.
The morphology of the screened QAU-Tn-E-7 cells is mostly short fusiform, and the cell size is 43.5 +/-7.3 mu m multiplied by 24.5 +/-4.1 mu m; the infection rate of autographa californica nuclear polyhedrosis virus (AcMNPV) in 24-well cell culture plates was 98.3%, and the average single-cell viral polyhedrosis yield was 115.8; the sequence analysis of the gene fragment of the CO I of the QAU-Tn-E-7 cell is 100 percent consistent with the fragment of the Co I of the Trichoplusia ni, and the cell line is derived from the Trichoplusia ni; RT-PCR detection proves that the cell line QAU-Tn-E-7 does not contain TNCL virus, overcomes potential risks in the production of medical drugs and vaccines, and has good application prospect.
QAU-Tn-E-7 cells were cultured in suspension in a suspension flask (spinner flash) at 2.0X 105Inoculating at cell/mL concentration, stirring at 100r/min to obtain the best cell growth state, and the cell concentration reaches the highest value at 6d to reach 4.29 × 106cell/mL, population doubling time 21.3 h.
The virus yield and the expression level of the recombinant protein of the cell line QAU-Tn-E-7 under the serum-free suspension culture condition in a suspension culture flask (spinner flash) are determined, and the virus yield is 3.12 multiplied by 108PIB/mL, beta-galactosidase expression reaches 3.61X 104IU/mL, the activity of alkaline phosphatase reaches 3.97 IU/mL.
The present invention will be described in detail with reference to examples.
EXAMPLE 1 establishment and screening of cell lines and biological Properties
1.1 establishment of cell lines
Collecting 100 eggs newly laid by Trichoplusia ni (Trichoplusia ni), sterilizing egg surface with 10% sodium hypochlorite and 70% ethanol for 5min in ultra-clean bench, washing with sterile water for 3 times, transferring to fine sieve with pore diameter of 100 μm, adding 5mL of Sf-900TMIII SFM Medium, grinding the egg grains with sterile rubber heads, transferring the cell filtrate to 25cm2The cells were cultured in a 28 ℃ incubator in a cell culture flask. And (3) periodically replacing a certain proportion of fresh culture medium according to the growth state of the cells, blowing the cells in a super-clean workbench to prepare cell suspension after the primary culture cells are grown and spread on the bottom of a culture bottle, and carrying out cell passage according to a certain proportion. After about 10 passages, the cell line is successfully established until the growth state of the cells is stable. The duration of 2 years is 53 cultures, and 9 strains of Trichoplusia ni embryo cell lines are successfully established. A cell line which is sensitive to baculovirus and grows in a suspension manner is finally obtained by screening through measuring and comparing a series of indexes such as cell morphology, growth speed, sensitivity to virus, virus polyhedron yield and the like of 9 cell lines, and is named as QAU-Tn-E-7; the preservation number is as follows: CCTCC NO of C201865; the preservation time is as follows: year 2018, month 4, day 17; the storage unit is: china center for type culture Collection.
1.2 morphological characteristics of cell lines
The morphology of the cells was observed under an inverted phase contrast microscope (Olympus IX71), 100 cells were randomly selected, the ratio of cells of different morphologies was counted, and the size of the cells was measured by microscaling. The cells of the cell line QAU-Tn-E-7 are frequently short fusiform and account for 95% or more of the total number of cells, and the size of the cells is 43.5. + -. 7.3. mu.m.times.24.5. + -. 4.1. mu.m (FIG. 1).
1.3 molecular characterization of cell lines
Mitochondrial cytochrome c oxidase I (cytochrome c oxidase I, COI) of the cell line is subjected to PCR amplification by using universal primers HCO 2198 and LCO 1490(Folmer et al, 1994), and PCR products are subjected to sequencing after being detected by electrophoresis. The length of the CO I gene fragment of the QAU-Tn-E-7 cell line is 633bp, and the maximum similarity rate of the fragment and the CO I gene fragment sequence of the Trichoplusia ni in NCBI (NCBI accession number: MF679178) is 100% through sequence comparison and analysis, thereby proving that the QAU-Tn-E-7 cell line is the Trichoplusia ni cell line.
1.4 detection of alpha-nodaviruses in cell lines
Total RNAs of cell lines QAU-Tn-E-7 and BTI5B1-4 were extracted using TransZol Up Plus RNA Kit (Beijing Alternal gold Biotechnology Co., Ltd.), and cDNA was synthesized by reverse transcription using TransScript First-Strand cDNA Synthesis SuperMix Kit (Beijing Alternal gold Biotechnology Co., Ltd.) using sterile water as a control, reference literature (Li T C, et al, tension infection of a new alphanodavirus in an infection cell line, Journal of virology,2007,81(20):10890-10896), and amplification detection of α -nodavirus 2 genome fragment using primers Noda-D4(5-ACATCCAGATCCGATCAAGT-3) and Noda-U4 (5-GCCAGGAATGTTGCTTGCAA-3). The PCR reaction conditions are as follows: denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45s for 30 cycles, and final extension at 72 ℃ for 7 min. The PCR amplification products were detected by agarose electrophoresis. The results are shown in FIG. 2, and a 690bp fragment was amplified from BTI5B1-4 cells, while a fragment of the corresponding size was not amplified from QAU-Tn-E-7 cells and water, demonstrating that the TNCL virus was not contained in QAU-Tn-E-7 cells.
1.5 cell line growth Curve assay
Taking QAU-Tn-E-7 and BTI-Tn5B1-4(High Five) cells in logarithmic growth phase, counting with a blood counting chamber, diluting to 2 × 10 with culture medium5cell/mL to 25cm2In each culture flask, 5mL of Sf-900 was usedTMIII SFM medium and 10% FBS TNM-FH medium at 28 degrees C under culture. 3 flasks of cells were taken daily, counted on a hemocytometer, cell concentration calculated, and cell population doubling time calculated. Equal amounts of cell suspension were mixed with 0.4% trypan blue at the same time and the viability of the cells was determined on different days of culture.
QAU-Tn-E-7 cells at 25cm2The growth curve in the culture flask (T-flash) is shown in FIG. 3, the growth is slow in 1d after inoculation, the cell survival rate is 92.3%, the growth speed is gradually increased, the cell number is exponentially increased, the cell survival rate is more than 95% after 2d in the logarithmic phase growth stage, the 6d cell reaches the highest concentration of 3.62X 106Cellsand/mL. Then, as the culture time was prolonged, the cell concentration began to gradually decrease. Calculated, the QAU-Tn-E-7 cells are in Sf-900TMIII SFM medium under the condition of 28 ℃ the population doubling time is 23.3h, and the growth speed is faster than that of BTI-Tn5B1-4(High Five) cells cultured by TNM-FH medium containing 10% FBS.
1.6 cell line viral susceptibility assay
Taking cells in logarithmic growth phase, according to the ratio of 2.0 × 105The concentration of cells/mL is inoculated into a 24-well cell culture plate, and each well is 1 mL; culturing at 28 deg.C for 2h to make the cells adhere to the wall, removing the culture medium by suction, inoculating Autographa californica nuclear polyhedrosis virus (AcMNPV) with the amount of virus with multiplicity of infection (MOI) of 10, setting 3 times of repetition, slowly shaking on a vertical shaking table, adsorbing for 2h, discarding the virus liquid, adding 1mL of fresh culture medium into each well, and culturing in an incubator at 28 deg.C. And (3) checking the virus infection result under an inverted microscope, taking a picture, taking virus polyhedra formed in cells as an infection index, randomly selecting three fields per hole, counting the total number of the cells and the number of infected cells, and calculating the infection rate of the virus. Cells were harvested, sonicated to release viral polyhedra, virus concentration was counted and the yield of viral polyhedra per cell was calculated.
Infection symptoms begin to appear 2d after the cells are inoculated with the virus, the cell nucleus begins to swell, polyhedron formation can be seen in the cell nucleus after 3d, infection symptoms are obvious after 4d, a large amount of polyhedron is generated in the cells (figure 4), the cells gradually begin to be broken, and polyhedron is released into a culture medium. The infection rate of 4d cells inoculated with the virus is determined to be 98.3%, and the average yield of virus polyhedra per cell reaches 115.8 PIB.
EXAMPLE 2 serum-free suspension culture of cell lines
QAU-Tn-E-7 cells were grown at 75cm2The cells were cultured in 15mL cells in a T-flash flask, and the cell suspension was taken out in the logarithmic growth phase and inoculated into 125mL suspension culture flasks (Spinner flash, Corning Co.) and inoculated into 80mL cells at a concentration of 2.0X 105cells/mL QAU-Tn-E-7 cells with Sf-900 as culture mediumTMIII SFM serum-free medium, placing in a 28 ℃ incubator at 80r/min,Performing suspension culture on cells at the rotating speeds of 100r/min and 120r/min, processing 3 bottles of cells at each rotating speed, respectively taking 1mL of cell suspension from each bottle every 24h, counting by using a blood counting chamber, and measuring the cell concentration; the viability of the different treated cells was determined by mixing equal amounts of cell suspension with 0.4% trypan blue.
The screening results of suspension culture at different rotating speeds show that the growth state of cells is best when stirring at a rotating speed of 100r/min, and the method is suitable for serum-free suspension culture of QAU-Tn-E-7 cells. The cell growth curve under the serum-free suspension culture condition is shown in figure 3, the survival rate of the cells after 2d culture is always over 95 percent, the cell concentration reaches the highest value at the 6 th day and reaches 4.29 multiplied by 106cells/mL, higher than 25cm2QAU-Tn-E-7 and BTI-Tn5B1-4(High Five) cell concentrations cultured in cell culture flasks. The population doubling time of the cells under the serum-free suspension culture condition is calculated to be 21.3h, which is more than 25cm2QAU-Tn-E-7 and BTI-Tn5B1-4(High Five) cultured in the cell culture flasks grew rapidly (FIG. 3).
Example 3 viral production of cell lines in serum-free suspension culture
QAU-Tn-E-7 cells in logarithmic growth phase were cultured at 2.0X 105cells/mL were inoculated into 125mL suspension flasks (Spinner flash, Corning Co.) containing 80mL of the culture medium Sf-900TMIII SFM serum-free medium, placing the medium in an incubator at 28 ℃, and performing suspension culture on cells at the rotating speed of 100 r/min. Culturing for 3d, sampling to detect cell concentration, inoculating Autographa californica nuclear polyhedrosis virus (AcMNPV) according to cell concentration and virus amount with multiplicity of infection (MOI) of 10, and repeating for 3 times. Viral infection 4d cell suspensions were taken and examined for viral infection rate under a microscope, cell suspensions were collected and lysed with ultrasound to lyse the cells to release polyhedra, the concentration of polyhedra was calculated with a hemocytometer, and the number of polyhedra produced per infected cell was counted on average.
The results show that the infection rate of the QAU-Tn-E-7 cells after being inoculated with AcMNPV 4d under the condition of serum-free suspension culture in a suspension culture bottle can reach 100 percent, and the average disease of each cellThe yield of virus polyhedra is 106.2, and the yield of virus polyhedra can reach 3.12 multiplied by 108PIB/mL。
Example 4 expression of recombinant proteins in cell lines in serum-free suspension culture
Three processes are set: QAU-Tn-E-7 cells were plated with Sf-900 in 125mL suspension flasksTMIII SFM medium culture, QAU-Tn-E-7 cells at 25cm2Sf-900 used in culture bottleTMIII SFM medium culture, BTI-Tn5B1-4(High Five) cells at 25cm2The recombinant viruses AcMNPV-beta-gal and AcMNPV-SEAP were cultured in a culture flask in TNM-FH medium containing 10% FBS, and the cultured cells were infected with each of the recombinant viruses AcMNPV-beta-gal and AcMNPV-SEAP at a rate such that the multiplicity of infection MOI was 10, respectively, and the cells were cultured at 28 ℃ and sampled every day. Centrifuging the sample at 5000rpm/min for 5min, crushing the cells with an ultrasonic crusher, centrifuging at 5000rpm/min for 5min after the cells are completely crushed, and taking the supernatant for later use. The expression levels of the recombinant proteins β -galactosidase (. beta. -gal) and alkaline phosphatase (SEAP) were determined by reference to the methods of Davis et al (1992) and Zheng et al (2005).
The expression results of beta-galactosidase by different culture methods are shown in FIG. 5, the expression level of beta-gal of the cells gradually increases with the number of culture days, and 6d QAU-Tn-E-7 cells are neutralized to 25cm in a 125mL suspension culture flask2Sf-900 used in culture bottleTMIII the expression quantity of beta-gal cultured in SFM medium reaches the highest and is respectively 3.61 multiplied by 104IU/mL and 3.08X 104IU/mL, the expression level of beta-gal in control BTI-Tn5B1-4 cells reached the highest level at 8 days (2.35X 10)4IU/mL). Wherein 6d, QAU-Tn-E-7 cells were inoculated in 125mL suspension flasks with Sf-900TMIII the beta-galactosidase expression quantity cultured by the SFM medium is the highest, and is respectively that QAU-Tn-E-7 cells are 25cm2Sf-900 used in culture bottleTMIII SFM Medium culture (3.08X 10)4IU/mL) and BTI-Tn5B1-4(High Five) cells at 25cm2Culture in culture flasks with TNM-FH medium containing 10% FBS (2.23X 10)4IU/mL) 1.17-fold and 1.62-fold of the expression amount.
The results of the expression of alkaline phosphatase in the cells by different culture methods are shown in FIG. 6, the SEAP expression level of the cells gradually increases with the number of days of culture, and 7d QAU-Tn-E-7 is culturedThe SEAP expression level of the cells in a 125mL suspension culture flask reaches the highest (3.97IU/mL), and the SEAP expression level is that of QAU-Tn-E-7 cells at 25cm2Sf-900 used in culture bottleTMIII SFM Medium (3.36IU/mL) and BTI-Tn5B1-4(High Five) cells at 25cm21.18-fold and 2.04-fold expression of TNM-FH medium (1.95IU/mL) containing 10% FBS was cultured in the culture flask. While QAU-Tn-E-7 cells were at 25cm2Sf-900 used in culture bottleTMIII SFM Medium culture and BTI-Tn5B1-4(High Five) cells at 25cm2Cells cultured in flasks with TNM-FH medium containing 10% FBS reached a maximum at 9d, of 3.47IU/mL and 2.06IU/mL, respectively.
Claims (8)
1. An insect cell line is characterized in that the insect cell line is a Trichoplusia ni (Trichoplusia ni) cell line QAU-Tn-E-7, and the preservation number is CCTCC NO: C201865.
2. Use of the insect cell line of claim 1 for propagating a virus.
3. The use of claim 2, wherein the virus is an insect baculovirus.
4. The use of claim 3, wherein the virus is Autographa californica nuclear polyhedrosis virus.
5. A method of propagating Autographa californica nuclear polyhedrosis virus, said method comprising propagating the virus using the insect cell line of claim 1.
6. Use of the insect cell line of claim 1 to express a foreign protein.
7. The use of claim 6, wherein the foreign protein is β -galactosidase.
8. The use of claim 6, wherein the foreign protein is alkaline phosphatase.
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