Reconstructed ST cell strain ST-CTTN-KD as well as construction method and application thereof

Abstract

The invention belongs to the field of cytobiology, particularly relates to the cross technical field of cytobiology and vaccine production, and more particularly relates to a reconstructed ST cell strain ST-CTTN-KD as well as a construction method and application thereof. The preparation method comprises the following steps: designing shRNA (short hairpin ribonucleic acid) aiming at a swine CTTN coding sequence XM021082649.1, inserting a pLV3 lentiviral vector to form a recombinant vector transfected cell, collecting lentivirus, and further infecting an ST cell, so as to obtain the recombinant ST cell ST-CTTN-KD with stable and low-expression CTTN. The recombinant ST cell can significantly improve the titer of proliferating the hog cholera virus, especially increase the exosome secretion amount of the virus infected cell, thereby providing an important basis for virus research and vaccine production.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, in particular to the interdisciplinary field of cell biology and vaccine production, and more specifically to a reconstructed ST cell line ST-CTTN-KD and a construction method and application thereof. Background Art

[0002] Classical swine fever (CSF) is an infectious disease caused by the classical swine fever virus (CSFV). After infecting host cells, CSFV not only produces progeny virions through conventional pathways but also utilizes the host cell's exosome system to secrete exosomes containing intact viral nucleic acid components. Exosomes can then enter healthy cells and generate infectious, complete virions.

[0003] Further studies have shown that treating purified exosomes with CSFV neutralizing antibodies does not prevent exosomes from entering ST cells. Instead, exosomes activate the innate immune system of ST cells, but the activation pattern and intensity differ from those caused by viral particle infection. Therefore, exosomes play an important role in viral secondary infection and non-lesional proliferation.

[0004] Cortactin (CTTN) is present in many cell types and plays a key role in actin cytoskeleton dynamics, cell migration, adhesion and intracellular trafficking. Summary of the Invention

[0005] The present invention aims at the current field of swine fever virus proliferation technology, especially the field of vaccine production technology based on this, and provides a reconstructed ST cell line ST-CTTN-KD, which is classified and named wild boar Sus scrofa. The cell line was deposited in the General Microbiology Center of China Culture Collection Administration on April 23, 2025, with the deposit number CGMCCNO.46351, and the deposit location is Beijing, China.

[0006] This cell line can achieve stable changes in CTTN transcription levels and, by designing CTTN shRNA, achieve CTTN protein knockdown in the target cell line.

[0007] At the same time, the present invention further discloses a shRNA targeting CTTN, the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2:

[0008] Furthermore, the present invention also discloses a method for constructing a reconstructed ST cell line ST-CTTN-KD, comprising the following steps: (1) Designing shRNA that effectively knocks down CTTN; specifically, designing shRNA against the porcine CTTN coding sequence XM_021082649.1 to obtain shRNA with CTTN knockdown effect for constructing vectors; (2) inserting the shRNA into a pLV3 lentiviral vector to construct a recombinant pLV3 lentiviral vector, transfecting cells with the recombinant pLV3 lentiviral vector, and harvesting the lentiviral solution; preferably, the sense strand and antisense strand of the shRNA are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; (3) Infect ST cells with lentiviral concentrate and continue culturing after infection; (4) Screen and collect surviving cells, expand and establish cell lines to obtain ST-CTTN-KD cells.

[0009] On the other hand, the present invention also discloses the use of the reconstructed ST cell line ST-CTTN-KD in virus propagation.

[0010] In particular, the use of the reconstructed ST cell line ST-CTTN-KD in the proliferation of classical swine fever virus.

[0011] In particular, the reconstructed ST cell line ST-CTTN-KD is used to propagate classical swine fever virus under high MOI conditions.

[0012] The results showed that ST-CTTN-KD reconstructed cells had a higher CSFV production than ST original cells, and the difference in virus proliferation titer became more obvious as the MOI increased.

[0013] On the other hand, the present invention also discloses the use of the reconstructed ST cell line ST-CTTN-KD in increasing the secretion of exosomes from virus-infected cells, in particular the use of the reconstructed ST cell line ST-CTTN-KD in increasing the secretion of exosomes from cells infected with classical swine fever virus.

[0014] The results showed that compared with the original ST cells and ST-CTTN-OE cells (CTTN overexpression), ST-CTTN-KD cells not only had an increase in the total amount of exosomes, but also the appearance time of Core protein in their contents was earlier, indicating that changes in the transcription level of CTTN affected both the exosome load and content composition.

[0015] The present invention designs shRNA targeting the porcine CTTN coding sequence XM_021082649.1. This shRNA is then inserted into the pLV3 lentiviral vector to form a recombinant vector for transfection into cells. The lentivirus is then collected and further infected into ST cells to produce recombinant ST cells (ST-CTTN-KD) that stably and underexpress CTTN. These recombinant ST cells significantly increase the titer of classical swine fever virus (CSFV), particularly by increasing the secretion of exosomes from virus-infected cells, providing an important foundation for virus research and vaccine production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the recombinant pLV3 lentiviral vector.

[0017] Figure 2 The figures show the results of immunoblotting (WB) of original ST cells, ST-CTTN-KD cells (CTTN knockdown strain) and ST-CTTN-OE (CTTN overexpression strain) at different culture times.

[0018] Figure 3 The figures show the results of immunoblotting (WB) of exosome marker proteins and Classical Swine Fever Virus Core protein in original ST cells, ST-CTTN-KD cells (CTTN knockdown strain) and ST-CTTN-OE (CTTN overexpression strain) infected with CSFV at different culture times.

[0019] Figure 4 Schematic diagram comparing the number of exosome particles in naive ST cells, ST-CTTN-KD cells (CTTN knockdown strain) and ST-CTTN-OE (CTTN overexpression strain) after classical swine fever virus infection.

[0020] Figure 5 Schematic diagram comparing the survival rates of original cells (Naïve ST) and ST-CTTN-KD cells (CTTN knockdown strain) infected with classical swine fever virus at different MOIs.

[0021] Figure 6 Schematic diagram of the comparison of viral titers of ST original cells and ST-CTTN-KD cells infected with classical swine fever virus at different MOIs.

[0022] Figure 7 This is a schematic diagram showing the comparison of the proliferation generations and CSFV titers of ST-CTTN-KD reconstructed cells and ST original cells with the same confluence when infected with classical swine fever virus at MOI=0.1 and MOI=0.6, respectively. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the present invention is further described below with reference to specific embodiments.

[0024] Unless otherwise specified, the instruments and devices used in the embodiments of the present invention are all commercially available products, and all operations can be performed with reference to relevant instructions in the art.

[0025] In the present invention, ST-CTTN-KD cells, ST-CTTN-KD, and CTTN knockdown strains have the same meanings, and all refer to cells with CTTN knockdown expression.

[0026] In the present invention, ST-CTTN-OE cells, ST-CTTN-OE, and CTTN overexpression strains have the same meanings, and all refer to cells overexpressing CTTN.

[0027] In the present invention, ST cells, ST original strains, original cells, and ST original cells have the same meaning and refer to original ST cells. Example 1

[0028] shRNA was designed against the porcine CTTN coding sequence XM_021082649.1. The shRNA sequence is as follows: Insert shRNA into pLV3 lentiviral vector to construct recombinant pLV3 lentiviral vector. The specific method is as follows: Synthesize the two primers described above, dissolve them in 20 μl of ddH₂O, mix thoroughly, heat at 95°C for 3 minutes, and then cool to anneal at room temperature. Clone the primer dimer into the pLV3 vector using the EcoRI and BamHI restriction enzyme sites. After transformation, isolate a single recombinant colony, extract the plasmid, verify with sequencing, and store at -20°C until ready for use.

[0029] Recombinant pLV3 lentiviral vector Figure 1 .

[0030] Transfect cells with the recombinant pLV3 lentiviral vector and harvest the lentiviral solution. The specific method is as follows: The recombinant pLV3 vector, pMD2.G, and pSPAX.Z plasmids were co-transfected into HEK293 cells. The supernatants were collected 48 and 72 hours after transfection and concentrated with lentivirus concentrate (Clontech, 631231) to obtain recombinant lentivirus.

[0031] ST cells were infected with lentiviral concentrate, and the cells were cultured after infection. Surviving cells were screened and collected to expand and establish a cell line to obtain ST-CTTN-KD cells. The specific method is as follows: Six to eight hours after ST cells were infected with the recombinant lentivirus, the culture supernatant was discarded, the cells were washed three times with PBS, and the culture medium was switched to DMEM supplemented with 10% FBS and cultured in a 5% CO2 incubator at 37°C. After 48 hours, the cells were digested and puromycin was added to the culture medium at a concentration of 5 μg / mL. After 6 to 8 hours of incubation, the supernatant was discarded, the cells were washed three times with PBS, and the cells were cultured in medium without puromycin. This selection process was repeated, gradually increasing the puromycin concentration to 20 μg / mL. The cells were then subcultured in standard culture medium. The cells were then harvested and the knockdown of CTTN protein was assessed by western blot. Example 2

[0032] According to the method in Example 1, a CTTN (Genebank: XM_021082649.1) overexpression vector was constructed to overexpress the recombinant CTTN protein fused with a His tag in ST cells to create the protein overexpression control strain ST-CTTN-OE cell line.

[0033] Western blot (WB) was used to detect the expression of CTTN in different cell lines. Figure 2 shown.

[0034] according to Figure 2 As can be seen, the intracellular CTTN concentration in the CTTN-overexpressing cell line remained significantly elevated compared to the original cells throughout the 72-hour culture period, and a high concentration of the His tag was detected. The intracellular CTTN concentration in the CTTN-knockdown cell line continued to decrease compared to the original cells. This result indicates that the ST cell line with stably altered CTTN transcription levels was successfully constructed. The two cell lines were designated ST-CTTN-OE (CTTN overexpression) and ST-CTTN-KD (CTTN knockdown). Example 3

[0035] Original ST cells, ST-CTTN-KD cells, and ST-CTTN-OE cells were infected with CSFV, and exosome marker proteins and CSFV Core protein were detected by immunoblotting (WB) to compare the exosome secretion amount of different cell lines and the content of CSFV Core protein in exosomes.

[0036] The experimental method is as follows: 72 hours after infection, the culture supernatant was collected and centrifuged at 500 × g, 2000 × g, and 10,000 × g for 30 minutes, respectively, using a tabletop centrifuge (ThermoFisher, Multifuge X3R) and filtered through a 0.22 μM filter (Sartorius, Minisart PES) to remove cell debris and large extracellular vesicles (LEVs). Exosomes were then pelleted using an ultracentrifuge (Beckman Coulter, Optima L-100XP) at 100,000 × g for 2 hours. The supernatant was discarded and the pellet was resuspended in PBS to obtain the ultracentrifuge pellet (UC-pellets). Protein A magnetic beads (Beyotime, P2102) were coated with an anti-E2 protein monoclonal antibody (CVCC, Z294), mixed with the ultracentrifuge pellet, and placed on a rotating reactor for 4 hr. The reaction was carried out at 400 °C overnight, and the supernatant was collected after the reaction was completed; the above-mentioned immunomagnetic bead purification operation was repeated three times, and the supernatant was finally collected to obtain purified exosomes.

[0037] The results are as follows Figure 3 As shown, WB analysis revealed that 24 hours after infection, the concentration of the exosome marker protein syntenin was low in all three cell lines due to the short infection time. From 48 to 72 hours, the concentration of syntenin in the exosomes of ST-CTTN-KD cells increased rapidly, while that of ST-naive cells and ST-CTTN-OE cells increased more slowly. The concentration of syntenin in ST-CTTN-KD exosomes differed significantly from that of the other two cell lines. The concentration trend of CSFV Core protein followed a similar pattern to that of syntenin, but with slight differences. At 24 hours post-infection, the concentration of Core protein in the exosomes of ST-CTTN-KD cells was significantly higher than that of the other two cell lines. From 48 to 72 hours post-infection, the exosomes of ST-CTTN-KD cells consistently contained high concentrations of Core protein, significantly higher than those of ST-naive cells and ST-CTTN-OE cells. The results showed that compared with the other two cell lines, ST-CTTN-KD cells not only had an increase in the total amount of exosomes, but also had an earlier appearance time of Core protein in their contents, indicating that changes in the transcription level of CTTN affected both the exosome load and the composition of the contents.

[0038] Furthermore, the specific loading amount in exosomes was detected by nanoparticle tracking analyzer (NTA). Figure 4 As shown, combined Figure 4It can be seen that 24 hours after infection, the total amount of exosome secretion of ST-CTTN-OE cells was significantly lower than that of the other two strains, while the exosome secretion of ST-CTTN-KD cells was slightly higher than that of the original cell strain, but the difference was not significant; 48 hours after infection, the difference in exosome load gradually became significant, with ST-CTTN-OE cells significantly lower than the ST original strain, and the ST original strain significantly lower than ST-CTTN-KD cells; 72 hours after infection, the exosome secretion of ST-CTTN-KD cells was still significantly higher than that of the other two cell strains, while the exosome load of the ST original strain and ST-CTTN-OE was close. Example 4

[0039] Original ST cells and ST-CTTN-KD cells were infected with CSFV at different multiplicities of infection (MOI = 0.3, 0.6, and 1.0) to compare cell viability. Cell viability and viral titer were determined by trypan blue staining.

[0040] The results are as follows Figure 5 As shown in the figure, it can be seen that at MOI = 0.3, the survival rate of ST-CTTN-KD cells was always higher than that of original ST cells, but the difference between the two was not significant throughout the whole process; when the MOI increased to 0.6, the survival rate of original ST cells decreased rapidly 48 hours after infection, and the survival rates at 60 and 72 hours were significantly lower than those of ST-CTTN-KD cells; when the MOI increased to 1.0, the survival rates of both groups of cells decreased further, and the cell activity of original ST cells decreased rapidly 48 hours after infection and was significantly lower than that of ST-CTTN-KD cells, and the difference further widened at 72 hours.

[0041] Further, combined with Figure 6 It can be seen that in the MOI gradient experiment, the virus titer and biological characteristics of the original ST cells and ST-CTTN-KD cells showed significant differences. The original ST cells reached a peak titer (6.5lgTCID) at a low MOI (0.1). 50 However, the titer continued to decrease with the increase of MOI, reaching only 5.6 lgTCID at MOI = 1.0. 50 / mL, indicating its sensitivity to high multiplicity of infection. In contrast, the titer of ST-CTTN-KD cells reached a peak at MOI=0.6 (7.1lgTCID 50 / mL), which was significantly higher than the titer of the original strain at the same MOI (6.0 lgTCID 50 / mL), and still maintained 6.81 gTCID at MOI = 1.0 50 / mL, showing significant high MOI tolerance. Calculations show that when the MOI is 0.6, the virus yield of ST-CTTN-KD cells is 12.59 times higher than that of the original strain. Example 5

[0042] The ST-CTTN-KD reconstructed cells and ST original cells with the same confluence were infected with CSFV at MOI = 0.1 and MOI = 0.6, respectively. The culture supernatant was harvested every 3 days and the virus titer was tested. Figure 7 As shown in Figures A and B, at an MOI of 0.1, ST-CTTN-KD reconstructed cells supported CSFV proliferation for 10 passages, while ST original cells only supported CSFV proliferation for 7 passages. Starting from the second passage, the CSFV titer of ST-CTTN-KD reconstructed cells was higher than that of the original cells. At an MOI of 0.6, ST-CTTN-KD reconstructed cells supported CSFV proliferation for 8 passages, while ST original cells only supported CSFV proliferation for 3 passages. Furthermore, the CSFV titer of ST-CTTN-KD reconstructed cells was consistently higher than that of the original cells. This indicates that ST-CTTN-KD reconstructed cells can enhance CSFV proliferation at different MOI conditions.

[0043] The above is a specific embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. The reconstructed ST cell line ST-CTTN-KD was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on April 23, 2025, with the deposit number CGMCC NO.46351.

2. shRNA targeting CTTN as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. The method for constructing the reconstructed ST cell line ST-CTTN-KD according to claim 1, characterized in that: The following steps are involved: (1) Design of shRNA to effectively knock down CTTN; (2) Inserting shRNA into the pLV3 lentiviral vector to construct a recombinant pLV3 lentiviral vector, transfecting cells with the recombinant pLV3 lentiviral vector, and harvesting the lentiviral solution; (3) Infect ST cells with lentiviral concentrate and continue culturing after infection; (4) Screen and collect surviving cells and expand them to obtain ST-CTTN-KD cells.

4. The method for constructing the reconstructed ST cell line ST-CTTN-KD according to claim 3, characterized in that: In step (1), shRNA was designed and screened using the porcine CTTN coding sequence XM_021082649.

1.

5. The method for constructing the reconstructed ST cell line ST-CTTN-KD according to claim 4, wherein: The nucleotide sequences of the shRNAs are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

6. Use of the reconstructed ST cell line ST-CTTN-KD according to claim 1 in virus propagation.

7. The use according to claim 6, characterized in that: The application of the reconstructed ST cell line ST-CTTN-KD in the proliferation of classical swine fever virus.

8. The use according to claim 7, characterized in that: The reconstructed ST cell line ST-CTTN-KD is used to propagate classical swine fever virus under high MOI conditions.

9. Use of the reconstructed ST cell line ST-CTTN-KD according to claim 1 in increasing the secretion of exosomes in virus-infected cells.

10. The use according to claim 9, characterized in that: The use of the reconstructed ST cell line ST-CTTN-KD in increasing the secretion of exosomes in cells infected with classical swine fever virus.

Images