| Porcine circovirus(PCV) taxonomically belongs to family circovirus genus circovirus and is one of the smallest animal virus found so far. The porcine circovirus contains a covalently closed single-stranded circular negative-strand DNA and has a genome size about 1.76 Kb. There are two known PCV serotypes, that is, PCV1 and PCV2.The PCV1 is non-pathogenic virus, while the PCV2 is a pathogeny of various common diseases. The infection of the PCV2 leads to immune suppression and the PCV2 and other pathogens together will cause mixed infection or secondary infection. In addition, the virus infection is persistent and enables the diseases to exist for a long time and to be difficult to be eradicated, causing great losses to the pig industry. At present, the control of the virus mainly uses PCV2 vaccine injection and strengthened production management. At the same time, with the development of molecular biology techniques and new molecular breeding techniques, the anti-PCV2 pigs become possible. Our laboratory used the RNA interference technology combined with cloning transgenic technology to successfully breed transgenic anti-PCV2 pig at F0 generation and the resistance was verified at the molecular level and cell level. In this study, transgenic boars at F1 generation and wild-type sows were hybridized to obtain anti-PCV2 transgenic pigs at F2 generation as objects. Genetic expression stability and anti-PCV2 infection effect detection and analysis were conducted from the DNA level, RNA level and in vivo level respectively to obtain a safe and effective anti-PCV2 transgenic pig new variety. The main results were as follows:(1) According to the biological fluorescent lamps and PCR testing, there were 26 transgenic pigs in 37 pigs in F2 generation with a positive rate was 70.2%, suggesting the exogenous genes can be stably inherited.(2) si RNA can be specifically expressed in transgenic pigs and the si RNA expression was significant difference in the same tissue of different individuals(P < 0.01).(3) The method of using PCV2 and immunostimulant can cause PMWS symptoms in piglet by poisoning attack. When the serum virus number was relative low, the transgenic pigs had some antiviral effect.(4) After poisoning attack, the antibody levels in the four groups decreased first and rose later. The rebounding speed was faster in the group A and group C than that in the group B and group D, respectively. In addition, in the day 56, the antibody levels were significantly higher in group C than in group D.(5) The blood routine examination results showed that there were no significant differences in blood indexes between the group A, group B, group C and group D in each period(all P > 0.05). The results demonstrated the metabolism and functional regulation maintained basically the same in transgenic pigs and the wild-type pigs.(6) During the poisoning attack period, the weights of pigs at each period showed that no differences between the transgenic pigs and the wild-type pigs(all P > 0.05).(7) After the poisoning attack, piglet tissues and organs were collected to conduct immunehistochemistry experiments, the results showed no difference in the PCV2 protein deposition between the transgenic pigs and the wild-type pigs.In summary, the obtained transgenic pigs can specifically express si RNA inhibiting the expression of PCV2. After the poisoning attack in living body, the pig serum antibodies firstly decreased and then increased and when the number of the serum virus was low, the transgenic pigs had some certain antiviral effect. The blood routine examination results showed that the transfer of the exogenous genes into the pigs does not affect the metabolism and body functions. |
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