To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBacTM1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Objective To construct a Type 1 diabetes model in miniature pigs and explore postoperative care strategies for effectively prolonging the survival time of the model pigs.Methods Seven Banna miniature pigs were selected for pancreatectomy.Glucose,vitamins,and antibiotics were administered for 3-5 days after surgery to aid recovery.Blood glucose and urine glucose levels were measured twice a day in the morning and evening to adjust insulin supplementation accordingly.The model pigs were observed daily and records were kept,including orexis,psychosis,weakness,skin ulcer,and feces and urine.Body weight was measured weekly until the death of the model animals.Based on the model pigs'condition,glucose injection and Ringer's lactate solution were administered to supplement nutrition and correct electrolyte imbalances.Results All seven Banna miniature pigs showed typical symptoms of diabetes:random blood glucose levels higher than 11.1 mmol/L after pancreatectomy,far exceeding the average blood glucose level of 6.0 mmol/L in normal pigs;positive urine glucose;and progressive weight loss.These features indicated the successful construction of Type 1 diabetes model.Additionally,Type 1 diabetic pigs that survived more than 8 weeks showed progressive hair loss and skin ulceration.Euthanasia was performed on model pigs when they were unable to stand or even eat independently,and pathological examination and HE staining were conducted on tissues collected from affected organs such as the liver,kidneys,and skin.Pathological sections revealed liver congestion,massive glycogen accumulation,ballooning degeneration of hepatocytes,and progressive liver fibrosis,along with glomerular congestion,vacuolar degeneration in renal tubular epithelial cells,proteinuria,dermal congestion,thinning of vascular walls,and varying degrees of parakeratosis and dyskeratosis in the liver,kidneys,and skin tissues due to prolonged hyperglycemia.The average survival time of the constructed Banna miniature pig diabetes model was 44 d,with a maximum survival time of 121 d.Conclusion Type 1 diabetes model can be constructed successfully in Banna miniature pigs through pancreatectomy.With meticulous postoperative care,a long-term Type 1 diabetes model with significant complications can be achieved,providing a stable large-animal model for Type 1 diabetes treatment strategies.

Objective To establish a method for acquisition, perfusion, preservation and transportation of the genetically modified pig kidneys. Methods An eight genetically modified pig was utilized as experimental subject. Prior to kidneys procurement, the health status of the pig was assessed through hematology examination, and the vascular structure of the kidneys was examined using imaging techniques. Following kidneys acquisition, the pig kidneys were perfused and subsequently packaged into the cryogenic storage container labeled "For Organ Transportation Only" for interprovincial transport after communicating the transportation process with transportation department. To evaluate pathological damage to the pig kidneys, a serious of methods were employed such as hematoxylin-eosin (HE) staining, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorescence staining and enzyme-linked immune absorbent assay (ELISA). Results The preoperative examination of the eight genetically modified pig showed that the serum creatinine was 73.2 μmol/L, blood urea nitrogen was 2.8 mmol/L and hemoglobin was 116 g/L, all within the normal range, indicating normal renal function. CT angiography revealed no lesions in the pig kidneys, and no dilation, stenosis or premature branching of the blood vessels. The total time of obtaining the left and right kidneys from the eight genetically modified pig was (125 ± 10) min, with a blood loss of (20 ± 2) mL. The warm ischemia times were 3 min and 7 min, respectively. The perfusion and trimming times of the left and right kidneys were 36 min and 41 min, respectively. After perfusion, both kidneys were white and moist. The cold preservation and transportation time was 8 h. HE staining showed that some glomeruli were shrunk, and the lumens of the surrounding renal tubules were slightly depressed and swollen with partial inner membrane shedding and microvacuoles formed when the kidneys were preserved for 8 h. The level of cysteinyl aspartate-specific proteinase-3 messenger RNA in the kidneys tissue gradually increased with the extension of cold preservation time after 2 h (P<0.05). TUNEL fluorescence staining showed that only a small number of cells underwent apoptosis after 8 h of cold preservation, which was not significantly different from that at 0 h (P>0.05). ELISA results showed that the contents of lactate dehydrogenase (LDH) and creatinine in the preservation solution remained relatively stable, but the content of kidney injury molecule 1 (KIM-1) gradually increased with the extension of preservation time, suggesting that the pig kidneys had mild injury. Conclusions By establishing methods for acquisition, perfusion, preservation and transportation of the kidneys from genetically modified donor pig, it is possible to effectively and reliably use genetically modified pig kidneys for xenotransplantation.

ObjectiveXenotransplantation is an effective way to address the shortage of human organ donors, but it faces serious immune rejection reactions, including hyperacute rejection caused by blood type differences. Establishing a stable, convenient, and reliable method for pig blood type identification can quickly screen suitable donor pigs for xenograft research.MethodsBanna miniature inbred pigs, Diannan small eared pigs, and Bama Xiang pigs were selected as the research objects. DNA was extracted from the blood, oral buccal mucosa, and fetal fibroblasts of the three strains of pigs using DNA extraction kits. The target fragment of the ABO homologous gene EAA intron 7 in pigs was amplified using PCR method. Blood agglutination reaction was used to detect hemolysis in pig anterior vena cava whole blood after adding anti A and B antibodies. Immunohistochemical method was used to detect the expression level of A antigen in pig heart, liver, spleen, lung, and kidney tissues. Immunofluorescence method was used to detect the expression level of A antigen in pig oral mucosa. By comparing the results of different methods for determining pig blood types, the stability and reliability of the PCR method were verified, and a convenient PCR based pigblood type identification method was established.Results Firstly, the blood PCR results of 69 inbred strains of Banna miniature pigs, 7 Diannan small eared pigs, and 34 Bama Xiang pigs showed 20 AO blood types, 66 AA blood types, and 24 O blood types. The PCR results of fetal fibroblasts from 47 Diannan small eared pigs showed that all 47 fetuses were O blood type. Among them, the oral mucosal PCR results of 8 gene edited cloned pigs were consistent with those of donor fetal fibroblasts, all of which were O blood type. The oral mucosal PCR results of 8 wild-type pigs (2 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs) were consistent with the blood PCR identification results. Then, 11 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs were randomly selected for blood agglutination reaction validation, and the results were consistent with the PCR identification results of both blood samples and oral mucosa samples. Moreover, immuno-histochemical analysis was performed on the heart, liver, lung, kidney, and spleen tissues of one Banna miniature pig inbred line and two Bama Xiang pigs, and the results were consistent with blood PCR identification and blood agglutination reaction results. Finally, oral mucosal samples were collected from 2 inbred strains of Banna miniature pigs and 1 Bama Xiang pig for immunofluorescence detection, and the results were consistent with the blood PCR identification results.ConclusionBy collecting fetal cells and oral mucosal samples from live pigs for PCR detection, the blood type of pigs can be accurately and efficiently identified, providing a convenient method for blood type screening of xenograft donor pigs.