Establishment Of A Helicoverpa Armigera Embryonic Cell Line And Its Susceptibility To Recombinant Baculovirus AcMNPV-EGFP

Insect cell culture plays an important role in the application of Baculovirus Expression Victor System and in the development of all fields of medicine,agriculture,and biology.In this paper,A new bollworm embryo cell line was established with Helicoverpa armigera eggs.This experiment mainly studied the establishment of the cotton bollworm embryonic cell line,cell culture,biological characteristics and the use of insect cell line.The establishment of a new cell line of H.armigera not only provided a potential material for related experimental research in vitro,but also laid a foundation for the related research of insect cell line.The main findings are as follows:1.Establishment of H.armigera embryonic cell line Ha168Using a culture medium containing 10%TNM-FH,the embryo cells of H.armigera were cultured in vitro for approximately two years.Finally,a cell line with stable biological characteristics,good growth,and stable passage was established and designated as Ha168.2.Characteristics of H.armigera embryo cell line Ha168?1?Cell morphological characteristics:Ha168 cell line is an adherent growth type cell,mainly round,spindle cells,and a small number of giant cells,of which round cells account for about 77%,the size is 14.30±2.804?m,n=100;spindle cells approximately 20%,size12.04±2.928×47.09±12.674?m,n=100.?2?Identification of Ha168 cell line:Comparing cytochrome c oxidase subunit I?COI?sequences of Sf9 cells,H.armigera eggs,and Ha168 cells The results showed that the COI gene sequence of Ha168 and H.armigera eggs exhibited 100%identity,the similarity to the COI gene sequence of Sf9 cells was only 91%.Ha168 and H.armigera eggs were similar in the bands amplified by DAF?DNA Amplification Fingerprinting?and RAPD?Random Amplified Polymorphic DNA?and were significantly different from Sf9.All the above results showed that the Ha168 cell line originated from the cotton bollworm tissue.?3?Growth analysis:The growth curve of Ha168 cell line was plotted,and the population doubling time?PDT?of Ha168 cell line at 25 generations was calculated to be56.8 h.?4?Karyotype analysis:It was found that the suitable concentration of hypotonic KCl solution was 0.7%.It was observed under a 1000-fold light microscope that the chromosomes of the H.armigera embryonic cells clustered in a spot-like manner in the nuclear region.Chromosome number distribution varied widely range from 20 to 210 with an average mode of 71.Observed under a 1000-fold light microscope,the chromosomes of H.armigera embryonic cells clustered in a spot-like manner in the nuclear region,and the chromosome morphology could not be identified.Only the number of chromosomes was counted?Fig.2-10?.The results showed a normal distribution and chromosome number.The distribution range is 20 to 210,with an average of 71.?5?Cryopreservation and thawing:The Ha168 cell line that was frozen in liquid nitrogen for 6 months was resuscitated,and it was observed that it could grow normally,indicating that this cell line can be stored in liquid nitrogen for a long period of time.?6?Mycoplasma detection:PCR detection Ha168 cell line,the result is no mycoplasma contamination.3.Expression level in Ha168 cell line?1?Susceptibility of Ha168 cell line to AcMNPV-EGFP:Ha168 cell line was infected with the recombinant virus AcMNPV-EGFP,and Sf9 cell line was used as a control.Half-tissue culture Infectious dose(Median Tissue Culture Infectious Dose,TCID₅₀)of AcMNPV-EGFP in Ha168 and Sf9 cell lines was measured using a limiting dilution method.,the results showed that:virus infection rate of Ha168 cell line is slower than Sf9,TCID₅₀0 of Ha168 is 10^-2.78,TCID₅₀0 of Sf9 is 10^-6.1,indicating the susceptibility of Sf9 is more than Ha168 cell line.?2?Expression level of EGFP:The protein expression level of Ha168 cell line was approximately 7/10 of Sf9.In summary,we established a new H.armigera cell line with cotton boll worm eggs,and conducted basic biological assays,virus susceptibility,and the determination of the expression level of foreign proteins,which has further research value.