Prrx Gene Family Of Oryctolagus Cuniculus: Cloning, Prokaryotic Expression And Expression Pattern

Prrx1(MHox, k2, Pmx, Prrx1), Prrx2(S8, Prx2) and Prrx3(Prx3, SHOX2, SHOT,OG12)are belong to highly conservative Homeobox gene family. The Prrx1 null mice have a cleft palate and mild hypoplasia of both the mandible and the zeugopodal bones of the limbs. In contrast, the Prrx2 null mice are morphologically normal, but have mild cardiac functional deficits. The phenotype of the double mutant mice showed that Prrx1 and Prrx2 each play a role in spine, hyoid arch, craniofacial, aortic arch, hypophysis and limb development. Some amphibians can regenerate injured body parts, even an amputated limb, without cicatrix. This perfect healing process may concern Prrx1 gene. For mammal, the gene that contributes to fetal scarless healing process is Prrx2 gene. Prrx3 gene expresses at mesoderm and ectoderm embryo, and this gene plays a role in cerebrum, spinal cord, nerves heart, craniofacial and proximal limb development. Methylation of Prrx3 is closely related with incidence of lung cancer, which makes itself an initial screening method of lung cancer. The objective of this study is to clone and analyze the structure of Prrx1, Prrx2 and Prrx3 genes, and investigate their expression patterns in Oryctolagus cuniculus, which could provide some valuable references for further study for these genes.This study mainly achieved the following achievements:1. Prrx1a(Accession number: KP726284), Prrx1b(Accession number: KM222500.1), Prrx2 and Prrx3(Accession number: KP726285)were correctly cloned. Their DNA and amino acid sequences were anlysised by softwares.2. pET32a(+)-Prrx1a/Prrx1b/Prrx2/Prrx3 recombinant plasmids were constructed and ransferred into the expression host E.coli BL21. Expression of fusion proteins were induced by IPTG, and expression conditions, such as IPTG concentration, time and temperature were optimized. Ultrasonication of E. coli BL2 and subsequently HistrapTM HP purification was performed. Good antigenicity of fusion proteins were confirmed by SDS –PAGE and Western blot.3. This article have developed a ranked profile of the top performing 6 reference genes(GAPDH, HPRT1, 18 S r RNA, B2 M, Sdha, β-actin) by stable expression in different development periods and tissues in Oryctolagus cuniculus. This study confirmed that the most stable reference genes were different in different periods and tissues of New Zealand white rabbit. To ensure minimal variation between individuals of an experimental group and the accurate statistical analysis of small fold changes, we recommend normalizing data to the geometric mean of at least 3 validated reference genes as above when related New Zealand white rabbit trials were performed.4. Expression patterns of Prrx1, Prrx1 a, Prrx1 b, Prrx2 and Prrx3 m RNA were investigated by RT-PCR in different organs and stages in Oryctolagus cuniculus. Expression of two transforms of Prrx1, Prrx1 a and Prrx1 b, were compare in partial organs.5. Expressions of Prrx1, Prrx2 and Prrx3 proteins were confirmed in different organs and stages in Oryctolagus cuniculus by immunohistochemistry. Expressions of Prrx1, Prrx2 and Prrx3 proteins were inconformity with m RNA because of post-transcriptional control. Nevertheless, a detailed localization of Prrx1, Prrx2 and Prrx3 was not carried out because of a lack of specific antibodies for each factor.