The Expression And Distribution Of ZIP12 Gene In Broiler Chickens With Ascites Syndrome

Broiler ascites syndrome?AS?is a nutritional metabolic disease that occurs in rapidly growing broilers and is characterized by right heart failure and massive ascites accumulation in the abdominal cavity.From a histopathological perspective,pulmonary vascular remodeling?PVR?and its associated pulmonary hypertension?PH?are the basis of broiler ascites syndrome.Research has shown that zinc transporter?ZIP12?plays a key regulatory role in pulmonary vascular remodeling in hypoxic mice,the development of PVR and PH was reduced by inhibition of ZIP12 expression,suggest that ZIP12 may be an important regulator of pulmonary hypertension.The key link in broiler ascites syndrome is pulmonary hypertension,and whether ZIP12 has a similar role in the pathogenesis of broiler ascites syndrome requires further study.A model of broiler ascites syndrome was successfully prepared by Intravenous CM-32 ion exchange cellulose suspension injection.The relative expression of ZIP12 in preascites and ascites broiler chicken tissues was detected by RT-PCR and immunohistochemistry,then the difference between them was compared.The research results prove that the ZIP12 gene play an important role in the development of broiler ascites syndrome,this discovery provides scientific basic data for the development of a new prevention and control method for the disease.Details are as follow:1.The construction of broiler ascites syndrome model120 one-day-old broilers were randomly divided into a test group?TG??n=90?and a control group?CG??n=30?.All broilers were allowed to have free access to the same diet ad libitum and had a 24h fluorescent illumination per day throughout the trial period.At 20 days old,the chickens of TG group were intravenously injected with ion-exchange cellulose CM-32 suspension.Broilers in CG group were injected intravenously with the same volume of physiological saline.At 42 days old,heart and blood of all chickens were taken,and autopsy results were observed,the right ventricle?RV?weight,the total ventricle?TV?weight,RV/TV ratio,AST and CK of broilers were measured.The results showed that the TG had obvious ascites and right ventricular hypertrophy.At the same time,compared with the CG,the m_rv/m_tv,AST and CK of the TG were significantly increased and m_rv/m_tv?0.3.The results show that the model is stable and consistent with the results obtained by the method of injecting cellulose particles in worldwide.2.Amplification of ZIP12 gene and sequence analysisThe chicken ZIP12 gene was amplified by RT-PCR and the sequence was deposited in GenBank?accession no.:KU178951?.Sequence analysis revealed that the ZIP12 gene was conserved within the genus and differences between species.It encodes 613 amino acids and has a protein size of 69 KD,multiple potential transmembrane regions exist,contains signal peptides.3.Prokaryotic expression of ZIP12 and preparation of its polyclonal antibodySpecific primers with double restriction sites for the ZIP12 gene sequence were designed and the amplified gene fragment was linked to pGEX-6P-1 prokaryotic expression plasmid.The constructed expression plasmid was transformed into expression bacteria BL21?DE3?,and the recombinant protein was successfully expressed by IPTG induction.The purified protein was mixed with adjuvant to immunize New Zealand white rabbits to prepare rabbit anti-ZIP12 polyclonal antibody.Finally,the rabbit serum was detected by Western-Bolt and ELISA,the results show that the polyclonal antibody has high titer and purity.4.The expression and distribution of ZIP12 in broiler chickens with ascites syndromeThe mRNA relative expression level of ZIP12 in heart,liver,spleen,lung and kidney in AS group,preascites?PAS?group and control group were detected by real-time quantitative PCR.Statistic analysis showed that relative expression level of ZIP12 in the liver and lung in AS and PAS groups was significant higher?P<0.01?than that in control.The result of immunohistochemistry showed that ZIP12 was widely distributed in various tissues and average integrated optical density?IOD?analysis showed that the expression level of ZIP12protein was significantly increased compared to the control group.These results indicate that ZIP12 is involved in the pathogenesis of broiler AS.