The Gene Mutation And Protein Expression Study On Mismatch Repair Gene MLH1in Marek’s Disease

Marek’s disease (MD) is a malignant lymphoma disease caused by Marek’s disease virus (MDV), which is a common infectious disease of chickens. MDV infection can lead to hosts’ immune suppression. It is the most economically significant diseases in the poultry industry for a long period of time no matter now or in the future. Mismatch repair genes (MMR) are responsible for correcting the bases mismatch in cells. Their defections leading to tumor-associated genes’ mutations can not be timely and effectively corrected, allowing tumor susceptibility. MLH1and MSH2genes are the most important members in the MMR family. When they are inactivated the DNA bases’ conversion and transversion can not be identified and repaired, making some of the tumor suppressor genes be inactive d or some tumor genes be activated, ultimately resulting in cells apotheosis or tumors formation. Under normal physiological conditions, MLH1and MSH2genes can express plentiful proteins to maintain the stability of the genes. However, when these genes have some mutations, including DNA bases changes, deletions or other abnormal changes, their normal expression can be affected, which appears mismatch repair dysfunction, thus leading to tumorigenesis. In this trail, we focused on the MLH1gene which is a important member of MMR family. We researched MD chicken’s MLH1gene’s mutations condition and its protein expression condition in the chickens’MD tumor tissues.The experiment was divided into two parts, the first part was to detect the MLH1gene’s19exons mutation condition. The specific operations were as follows:first we used the HE staining to determined the chickens’ MD tumor tissue and normal tissue and found the focies of infectious. Then we extracted DNA from them. The MLH1gene’s19exons’ sequences which had been published on the National Center for Biotechnology Information(NCBI) were used to designed primers through the software primer5.0. After that we ran the PCR using the DNA and the primers as materials. We used the electrophoresis to confirm the DNA bands that were we need. we used the PCR-SSCP technique to see the bands’ difference between the muscle tissue and the viscera tissue. From the pathological sections result, we exacted the DNA from the liver tumor and muscle tissue of the NO.2chicken, and ran the PCR using the19primers and got all the single band. In order to recycle the maximum amount DNA, we expanded the PCR system. We recycled all the purposed DNA bands and sent all the products to Shanghai company for sequencing.The Normal tissue and tumor tissue DNA sequencing results were analyzed and compared by the matching software to identify the nucleotide and amino acid differences, to find out and determined the exact location of the gene mutation, thus we could know whether the exon had mutation or not.The second part was to detect MLH1gene protein expression conditions in the MD tumor tissue with immunohistochemical(IHC) staining methods. We used the paraffin sections which had been done from formalin-fixed tumor tissue to do (hematoxylin and eosin) HE and immunohistochemistry (IHC and the T/B lymphocytes) staining. Through these ways we could find how the MLH1gene protein expression between tumor cells in the foci and normal cells not in the foci, in addition, we could tell the target tumor cell’s type.The PCR-SSCP results showed no difference between the muscle and tumor tissues about the same chicken.Through HE staining, we found that there were lots of nodular lesions on the NO.2chicken’s liver tissue. Then we extracted the liver tissue’s DNA and ran the PCRS with19pairs primers and sequencing by the company. The sequencing results received and analyzed by software. The analyzed results showed that the MLH1gene’s DNA had no changes among19exons. The second part result was that the NO.6chicken’s kidney MLH1protein’s expression was detected by immunohistochemical method. The MLH1protein expression conditions in the kidney’s tumor cells were different from the adjacent normal cells. The normal cells’ cytoplasmic MLH1gene express positively, but in the tumor lesions cell MLH1gene expressed lowly or not expressed at all. T/B lymphocytes Immunohistochemical method were used to find out the exact type of the tumor cells. Whether the tumor cell were B or T lymphocyte. Through that method, we found that the MD tumors’s cells were stained in black color, showed which expressed CD3antigen. It means that the kidney’ tissue tumor cell were T lymphocytes. The final results showed that maybe the MD tumor was T lymphocytes tumor.